Methods of imaging using osmotically stabilized microbubble preparations

ABSTRACT

A microbubble preparation formed of a plurality of microbubbles comprising a first gas and a second gas surrounded by a membrane such as a surfactant, wherein the first gas and the second gas are present in a molar ratio of from about 1:100 to about 1000:1, and wherein the first gas has a vapor pressure of at least about (760−x) mm Hg at 37° C., where x is the vapor pressure of the second gas at 37° C., and wherein the vapor pressure of each of the first and second gases is greater than about 75 mm Hg at 37° C.; also disclosed are methods for preparing microbubble compositions, including compositions that rapidly shrink from a first average diameter to a second average diameter less than about 75% of the first average diameter and are stabilized at the second average diameter; methods and kits for preparing microbubbles; and methods for using such microbubbles as contrast agents.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 08/480,853, filedJun. 7, 1995 and now U.S. Pat. No. 5,626,833, which is a division ofU.S. Ser. No. 08/284,083, filed Aug. 1, 1994 and now U.S. Pat. No.5,605,673, which is a continuation-in-part of U.S. Ser. No. 08/099,951filed Jul. 30, 1993, now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention includes a method for preparing stable long-livedmicrobubbles for ultrasound contrast enhancement and other uses, and tocompositions of the bubbles so prepared.

2. Background of the Art

Ultrasound technology provides an important and more economicalalternative to imaging techniques which use ionizing radiation. Whilenumerous conventional imaging technologies are available, e.g., magneticresonance imaging (MRI), computerized tomography (CT), and positronemission tomography (PET), each of these techniques use extremelyexpensive equipment. Moreover, CT and PET utilize ionizing radiation.Unlike these techniques, ultrasound imaging equipment is relativelyinexpensive. Moreover, ultrasound imaging does not use ionizingradiation.

Ultrasound imaging makes use of differences in tissue density andcomposition that affect the reflection of sound waves by those tissues.Images are especially sharp where there are distinct variations intissue density or compressibility, such as at tissue interfaces.Interfaces between solid tissues, the skeletal system, and variousorgans and/or tumors are readily imaged with ultrasound.

Accordingly, in many imaging applications ultrasound performs suitablywithout use of contrast enhancement agents; however, for otherapplications, such as visualization of flowing blood, there have beenongoing efforts to develop such agents to provide contrast enhancement.One particularly significant application for such contrast agents is inthe area of perfusion imaging. Such ultrasound contrast agents couldimprove imaging of flowing blood in the heart muscle, kidneys, liver,and other tissues. This, in turn, would facilitate research, diagnosis,surgery, and therapy related to the imaged tissues. A blood poolcontrast agent would also allow imaging on the basis of blood content(e.g., tumors and inflamed tissues) and would aid in the visualizationof the placenta and fetus by enhancing only the maternal circulation.

A variety of ultrasound contrast enhancement agents have been proposed.The most successful agents have generally consisted of microbubbles thatcan be injected intravenously. In their simplest embodiment,microbubbles are miniature bubbles containing a gas, such as air, andare formed through the use of foaming agents, surfactants, orencapsulating agents. The microbubbles then provide a physical object inthe flowing blood that is of a different density and a much highercompressibility than the surrounding fluid tissue and blood. As aresult, these microbubbles can easily be imaged with ultrasound.

Most microbubble compositions have failed, however, to provide contrastenhancement that lasts even a few seconds, let alone minutes, ofcontrast enhancement. This greatly limits their usefulness. Microbubbleshave therefore been “constructed” in various manners in an attempt toincrease their effective contrast enhancement life. Various avenues havebeen pursued: use of different surfactants or foaming agents; use ofgelatins or albumin microspheres that are initially formed in liquidsuspension, and which entrap gas during solidification; and liposomeformation. Each of these attempts, in theory, should act to createstronger bubble structures. However, the entrapped gases (typically air,CO₂, and the like) are under increased pressure in the bubble due to thesurface tension of the surrounding surfactant, as described by theLaplace equation (ΔP=2γ/r).

This increased pressure, in turn, results in rapid shrinkage anddisappearance of the bubble as the gas moves from a high pressure area(in the bubble) to a lower pressure environment (in either thesurrounding liquid which is not saturated with gas at this elevatedpressure, or into a larger diameter, lower pressure bubble).

Solid phase shells that encapsulate gases have generally proven toofragile or too permeable to the gas to have satisfactory in vivo life.Furthermore, thick shells (e.g., albumin, sugar, or other viscousmaterials) reduce the compressibility of the bubbles, thereby reducingtheir echogenicity during the short time they can exist. Solid particlesor liquid emulsion droplets that evolve gas or boil when injected posethe danger of supersaturating the blood with the gas or vapor. This willlead to a small number of large embolizing bubbles forming at the fewavailable nucleation sites rather than the intended large number ofsmall bubbles.

One proposal for dealing with such problems is outlined in Quay,PCT/US92/07250. Quay forms bubbles using gases selected on the basis ofbeing a gas at body temperature (37° C.) and having reduced watersolubility, higher density, and reduced gas diffusivity in solution incomparison to air. Although reduced water solubility and diffusivity canaffect the rate at which the gas leaves the bubble, numerous problemsremain with the Quay bubbles. Forming bubbles of sufficiently smalldiameter (e.g., 3-5 μm) requires high energy input. This is adisadvantage in that sophisticated bubble preparation systems must beprovided at the site of use. Moreover, The Quay gas selection criteriaare incorrect in that they fail to consider certain major causes ofbubble shrinkage, namely, the effects of bubble surface tension,surfactants and gas osmotic effects, and these errors result in theinclusion of certain unsuitable gases and the exclusion of certainoptimally suitable gases.

Accordingly, a need exists in the art for compositions, and a method toprepare such compositions, that provide, or utilize, a longer lifecontrast enhancement agent that is biocompatible, easily prepared, andprovides superior contrast enhancement in ultrasound imaging.

SUMMARY OF THE INVENTION

In accordance with the present invention, there is provided anultrasound contrast enhancement agent that has a prolonged longevity invivo, which consists of virtually any conventional microbubbleformulation in conjunction with an entrapped gas or gas, mixture that isselected based upon consideration of partial pressures of gases bothinside and outside of the bubble, and on the resulting differences ingas osmotic pressure that oppose bubble shrinkage. Gases having a lowvapor pressure and limited solubility in blood or serum (i.e.,relatively hydrophobic) may advantageously be provided in combinationwith another gas that is more rapidly exchanged with gases present innormal blood or serum. Surfactant families allowing the use of highermolecular weight gas osmotic agents, and improved methods of bubbleproduction are also disclosed.

One aspect of the present invention is a stabilized gas filledmicrobubble preparation, comprising a mixture of a first gas or gasesand a second gas or gases (gas osmotic agent) within generally sphericalmembranes to form microbubbles, wherein the first gas and the second gasare respectively present in a molar ratio of about 1:100 to about1000:1, and wherein the first gas has a vapor pressure of at least about(760−x) mm Hg at 37° C., where x is the vapor pressure of the second gasat 37° C., and wherein the vapor pressure of each of the first andsecond gases is greater than about 75 mm Hg at 37° C., with the provisothat the first gas and the second gas are not water vapor. In oneembodiment, the second gas comprises a fluorocarbon and the first gas isa nonfluorocarbon, such as nitrogen, oxygen, carbon dioxide, or amixture thereof.

The microbubbles may advantageously be provided in a liquid medium, suchas an aqueous medium, wherein they have a first average diameter, theratio of the first gas to the second gas in the microbubbles is at least1:1, and the microbubbles are adapted to shrink in the medium as aresult of loss of the first gas through the membrane to a second averagediameter of less than about 75% of the first diameter and then remainstabilized at or about the second diameter for at least about 1 minuteas a result of a gas osmotic pressure differential across the membrane.Advantageously, the medium is in a container and the microbubbles haveactually been formed in the container. Alternatively, the medium isblood in vivo. In one embodiment, the liquid medium contains gas orgases dissolved therein with a gas tension of at least about 700 mm Hg,wherein the first diameter is at least about 5 μm, and wherein thetension of the gas or gases dissolved in the medium is less than thepartial pressure of the same gas or gases inside the microbubbles.

In a particularly preferred embodiment, the bubble initially contains atleast three gases: a first gas having a partial pressure far greaterthan the gas tension of the same gas in the surrounding liquid (e.g.,1.5, 2, 4, 5, 10, 20, 50, or 100 or more times greater than in thesurrounding liquid); a second gas that is retained in the bubble due toa relatively low permeability of the bubble membrane to the gas, or arelatively low solubility of the gas in the surrounding medium (asdescribed elsewhere herein), and a third gas to which the membrane isrelatively permeable that is also present in the surrounding medium. Forexample, in an aqueous system exposed to or at least partiallyequilibrated with air (such as blood), the first gas may advantageouslybe carbon dioxide or another gas not present in large quantities in airor blood; the second gas may be a fluorocarbon gas, such asperfluorohexane; and the third gas may be air or a major component ofair such as nitrogen or oxygen.

Preferably, the first diameter prior to shrinkage is at least about 10μm and the second diameter at which the diameter is stabilized isbetween about 1 μm and 6 μm.

For all of the microbubble preparations or methods described herein, inone preferred embodiment, the second gas has an average molecular weightat least about 4 times that of the first gas. In another preferredembodiment, the second gas has a vapor pressure less than about 750 or760 mm Hg at 37° C. Moreover, it is preferred that the molar ratio ofthe first gas to the second gas is from about 1:10 to about 500:1,200:1, or 100:1. In other preferred embodiments, the second gascomprises a fluorocarbon or a mixture of at least two or threefluorocarbons, and the first gas is a nonfluorocarbon. In someadvantageous preparations, the second gas comprises one or moreperfluorocarbons. In others, both the first gas and the second gascomprise fluorocarbons. In still others, the microbubbles contain as thefirst gas, or as the second gas, or respectively as the first and secondgases, gaseous perfluorobutane and perfluorohexane in a ratio from about1:10 to about 10:1. Alternatively, the microbubbles contain as the firstgas, or as the second gas, or respectively as the first and secondgases, gaseous perfluorobutane and perfluoropentane in a ratio fromabout 1:10 to about 10:1. It is advantageous that the second gas leavethe microbubble much more slowly than does the first gas; thus, it ispreferred that the second gas has a water solubility of not more thanabout 0.5 mM at 25° C. and one atmosphere, and the first gas has a watersolubility at least about 10 times, and preferably at least 20, 50, 100,or 200 times greater than that of the second gas. Similarly, it ispreferred that the permeability of the membrane to the first gas is atleast about 5 times, preferably 10, 20, 50, or 100 times greater thanthe permeability of the membrane to the second gas.

The microbubble preparation may advantageously be contained in acontainer, having a liquid in the container in admixture with themicrobubbles, wherein the container further comprises means fortransmission of sufficient ultrasonic energy to the liquid to permitformation of the microbubbles by sonication. In this way, themicrobubbles can be formed by the physician (or other professional)immediately before use by applying ultrasonic energy from an outsidesource to the sterile preparation inside the container. This means fortransmission can, for example, be a flexible polymer material having athickness less than about 0.5 mm (which permits ready transmission ofultrasonic energy without overheating the membrane). Such membranes canbe prepared from such polymers as natural or synthetic rubber or otherelastomer, polytetrafluoroethylene, polyethylene terephthalate, and thelike.

In the microbubble preparations of the invention, the membrane enclosingthe gas is preferably a surfactant. One preferred type of surfactantcomprises a non-Newtonian viscoelastic surfactant, alone or incombination with another surfactant. Other preferred general andspecific categories of surfactants include carbohydrates, such aspolysaccharides, derivatized carbohydrates, such as fatty acid esters ofsugars such as sucrose (preferably sucrose stearate), and proteinaceoussurfactants including albumin. Alternatively, the membrane of themicrobubble need not be a fluid (such as a surfactant), but instead canbe a solid or semi-solid, such as hardened, thickened, or denaturedproteinaceous material (e.g. albumin), carbohydrates, and the like.

One advantageous form of the invention is a kit for use in preparingmicrobubbles, preferably at the site of use. This kit may comprisesealed container (such as a vial with a septum seal for easy removal ofthe microbubbles using a hypodermic syringe), a liquid in the container(such as water or a buffered, isotonic, sterile aqueous medium), asurfactant in the container, and a fluorocarbon gas (including afluorocarbon vapor) in the container, wherein the liquid, thesurfactant, and the fluorocarbon gas or vapor are together adapted toform microbubbles upon the application of energy thereto. The energyadvantageously may be simple shaking energy, either manual ormechanical, stirring or whipping, or ultrasonic energy. The kitpreferably includes means in the container for permitting transmissionof sufficient external ultrasonic energy to the liquid to formmicrobubbles in the container. As above, the means for transmission canin one embodiment comprise a flexible polymer membrane having athickness less than about 0.5 mm. In one embodiment, the kit furtherincludes a nonfluorocarbon gas in the container, wherein the molar ratioof the nonfluorocarbon gas to the fluorocarbon gas is from about 1:10 toabout 1000:1, with the proviso that the nonfluorocarbon gas is not watervapor. In all of the kits of the present invention, the surfactant, thegas or gases, and the other elements of the kit may in some embodimentsbe the same as recited above for the microbubble preparation per se.

In another embodiment, the kit comprises a container, driedliquid-soluble void-containing structures in the container, thevoid-containing structures defining a plurality of voids having anaverage diameter less than about 100 μm, a gas in the voids, and asurfactant, wherein the void-containing structures, the gas, and thesurfactant are together adapted to form microbubbles upon addition tothe container of a liquid in which the void-containing structures aresoluble. These void-containing structures can be made at least in partof the surfactant, e.g., by lyophilization of void-forming material orby spray drying, or can be formed from any other liquid soluble(preferably water soluble) film-forming material, such as albumin,enzymes, or other proteins, simple or complex carbohydrates orpolysaccharides, and the like. The surfactants used in the kit canadvantageously be those described above in connection with themicrobubble preparations per se.

The present invention also includes a method for forming microbubbles,comprising the steps of providing a first gas, a second gas, a membraneforming material, and a liquid, wherein the first gas and the second gasare present in a molar ratio of from about 1:100 to about 1,000:1, andwherein the first gas has a vapor pressure of at least about (760−x) mmHg at 37° C., where x is the vapor pressure of the second gas at 37° C.,and wherein the vapor pressure of each of the first and second gases isgreater than about 75 mm Hg at 37° C., with the proviso that the firstgas and the second gas are not water vapor, and surrounding the firstand second gases with the membrane forming material to form microbubblesin the liquid. The membrane forming materials and gases may be asdescribed above. The method preferably further comprises the steps ofinitially forming microbubbles having a first average diameter whereinthe initial ratio of the first gas to the second gas in the microbubblesis at least about 1:1, contacting the microbubbles having a firstaverage diameter with a liquid medium, shrinking the microbubbles in themedium as a result of loss of the first gas through the membrane, andthen stabilizing the microbubbles at a second average diameter of lessthan about 75% of the first diameter for a period of at least oneminute. Preferably, the microbubbles are stabilized at the seconddiameter by providing a gas osmotic pressure differential across themembrane such that the tension of a gas or gases dissolved in the mediumis greater than or equal to the pressure of the same gas or gases insidethe microbubbles. In one embodiment, the first diameter is at leastabout 5 μm.

The invention also includes a method for forming microbubbles,comprising the steps of providing dried liquid-soluble void-containingstructures, the void-containing structures defining a plurality of voidshaving a diameter less than about 100 μm, providing a gas in the voids,providing a surfactant, combining together the void-containingstructures, the gas, the surfactant, and a liquid in which thevoid-containing structures are soluble, and dissolving thevoid-containing structures in the liquid whereby the gas in theenclosures forms microbubbles that are surrounded by the surfactant. Aswith the kit, preferred void-containing structures are formed ofprotein, surfactant, carbohydrate, or any of the other materialsdescribed above.

Another aspect of the present invention is a method for producingmicrobubbles having increased in-vial stability, comprising the steps ofspray drying a liquid formulation of a biocompatible material to producehollow microspheres therefrom, permeating the microspheres with a gasosmotic agent (the second gas) mixed with the first gas and storing themicrospheres in a container with the gas mixture, and then adding anaqueous phase to the powder, wherein the powder dissolves in the aqueousphase to entrap the gas mixture within a liquid surfactant membrane toform microbubbles. In one embodiment, the microsphere comprises a starchor starch derivative having a molecular weight of greater than about500,000 or a dextrose equivalency value less than about 12. Onepreferred starch derivative is hydroxyethyl starch. Preferably, the gasosmotic agent comprises a perfluorocarbon. In another embodiment, themicrosphere comprises a sugar ester having a component with ahydrophilic-lipophilic balance less than about eight. Preferably, thesugar ester is sucrose tristearate.

The present invention also includes a method for forming microbubbles,comprising the step of providing a gas osmotic agent-permeatedsurfactant powder, and combining the powder with an aqueous phase.

Still another aspect of the present invention is a method for increasingthe in vivo half-life of microbubbles, comprising providing a spraydried formulation in combination with a gas osmotic agent that permeatesthe formulation, and combining the formulation with an aqueous phase toform microbubbles having a half-life in vivo of at least about 20seconds. In preferred embodiments, the spray dried formulation comprisesa starch or starch derivative and the sugar ester is sucrosetristearate.

Finally, the present invention includes a method for imaging an objector body, comprising the steps of introducing into the object or body anyof the aforementioned microbubble preparations and then imaging at leasta portion of the object or body via ultrasound or magnetic resonance.Preferably, the body is a vertebrate and the preparation is introducedinto the vasculature of the vertebrate. The method may further includepreparing the microbubbles in any of the aforementioned manners prior tointroduction into the animal.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a perspective view of a two chamber vial containing amicrobubble-forming preparation, with an aqueous solution in an upperchamber and solid and gaseous ingredients in a lower chamber.

FIG. 2 illustrates the vial of FIG. 1, where the aqueous solution hasbeen mixed with the solid ingredients to form microbubbles foradministration to a patient.

FIG. 3 is a perspective view of an inverted two-chamber vial containinga microbubble-forming preparation, with an aqueous solution in the lowerchamber and solid and gaseous ingredients in the upper chamber.

FIG. 4 illustrates the vial of FIG. 3, where the aqueous solution hasbeen mixed with the solid ingredients to form microbubbles foradministration to a patient.

DETAILED DESCRIPTION OF THE INVENTION

As used in the present description and claims, the terms “vapor” and“gas” are used interchangeably. Similarly, when referring to the tensionof dissolved gas in a liquid, the more familiar term “pressure” may beused interchangeably with “tension.” “Gas osmotic pressure” is morefully defined below, but in a simple approximation can be thought of asthe difference between the partial pressure of a gas inside amicrobubble and the pressure or tension of that gas (either in a gasphase or dissolved in a liquid phase) outside of the microbubble, whenthe microbubble membrane is permeable to the gas. More precisely, itrelates to differences in gas diffusion rates across a membrane. Theterm “membrane” is used to refer to the material surrounding or defininga microbubble, whether it be a surfactant, another film forming liquid,or a film forming solid or semisolid. “Microbubbles” are considered tobe bubbles having a diameter between about 0.5 and 300 μm, preferablyhaving a diameter no more than about 200, 100, or 50 μm, and forintravascular use, preferably not more than about 10, 8, 7, 6, or 5 μm(measured as average number weighted diameter of the microbubblecomposition). When referring to a “gas,” it will be understood thatmixtures of gases together having the requisite property fall within thedefinition, except where the context otherwise requires. Thus, air maytypically be considered a “gas” herein.

The present invention provides microbubbles that have a prolongedlongevity in vivo that are suitable for use as ultrasound and magneticresonance imaging (MRI) contrast enhancement agents. Typical ultrasoundcontrast enhancement agents exhibit contrast enhancement potential foronly about one pass through the arterial system, or a few seconds toabout a minute, and thus do not survive past the aorta in a patientfollowing intravenous injection. In comparison, contrast agents preparedin accordance with the present invention continue to demonstratecontrast enhancements lives measured in multiple passes through theentire circulatory system of a patient following intravenous injection.Bubble lives of several minutes are easily demonstrated. Suchlengthening of contrast enhancement potential during ultrasound ishighly advantageous. In addition, the contrast enhancement agents of theinvention provide superior imaging; for example, clear, vivid, anddistinct images of blood flowing through the heart, liver, and kidneysare achieved. Thus small, nontoxic doses can be administered in aperipheral vein and used to enhance images of the entire body.

As disclosed in U.S. Pat. No. 5,315,997, gases and perfluorocarbonvapors have magnetic susceptibilities substantially different fromtissues and blood. Therefore, the microbubbles of the present inventionwill cause changes in the local magnetic fields present in tissues andblood during MRI. These changes can be discerned on an MRI image and canbe used to detect the presence of a contrast agent. The agent of theinvention will persist in the blood pool longer and will therefore allowlonger, more sensitive scans of larger portions of the body.

While bubbles have been shown to be the most efficient ultrasoundscatterers for use in intravenous ultrasound contrast agents, their mainpractical drawback is the extremely short lifetime of the small(typically less than 5 microns diameter) bubbles required to passthrough capillaries in suspension. This short lifetime is caused by theincreased gas pressure inside the bubble, which results from the surfacetension forces acting on the bubble. This elevated internal pressureincreases as the diameter of the bubble is reduced. The increasedinternal gas pressure forces the gas inside the bubble to dissolve,resulting in bubble collapse as the gas is forced into solution. TheLaplace equation, ΔP=2γ/r, (where ΔP is the increased gas pressureinside the bubble, γ is the surface tension of the bubble film, and r isthe radius of the bubble) describes the pressure exerted on a gas bubbleby the surrounding bubble surface or film. The Laplace pressure isinversely proportional to the bubble radius; thus, as the bubbleshrinks, the Laplace pressure increases, increasing the rate ofdiffusion of gas out of the bubble and the rate of bubble shrinkage.

It was surprisingly discovered that gases and gas vapor mixtures whichcan exert a gas osmotic pressure opposing the Laplace pressure cangreatly retard the collapse of these small diameter bubbles. In general,the invention uses a primary modifier gas or mixture of gases thatdilute a gas osmotic agent to a partial pressure less than the gasosmotic agent's vapor pressure until the modifier gas will exchange withgases normally present in the external medium. The gas osmotic agent oragents are generally relatively hydrophobic and relatively bubblemembrane impermeable and also further possess the ability to develop gasosmotic pressures greater than 75 or 100 Torr at a relatively low vaporpressure.

The process of the invention is related to the well known osmotic effectobserved in a dialysis bag containing a solute that is substantiallymembrane impermeable (e.g. PEG, albumin, polysaccharide, starch)dissolved in an aqueous solution is exposed to a pure water externalphase. The solute inside the bag dilutes the water inside the bag andthus reduces the rate of water diffusion out of the bag relative to therate of pure water (full concentration) diffusion into the bag. The bagwill expand in volume until an equilibrium is established with anelevated internal pressure within the bag which increases the outwarddiffusional flux rate of water to balance the inward flux rate of thepure water. This pressure difference is the osmotic pressure between thesolutions.

In the above system, the internal pressure will slowly drop as thesolute slowly diffuses out of the bag, thus reducing the internal soluteconcentration. Other materials dissolved in the solution surrounding thebag will reduce this pressure further, and, if they are more effectiveor at a higher concentration, will shrink the bag.

It was observed that bubbles of air saturated with selectedperfluorocarbons grow rather than shrink when exposed to air dissolvedin a liquid due to the gas osmotic pressure exerted by theperfluorocarbon vapor. The perfluorocarbon vapor is relativelyimpermeable to the bubble film and thus remains inside the bubble. Theair inside the bubble is diluted by the perfluorocarbon, which acts toslow the air diffusion flux out of the bubble. This gas osmotic pressureis proportional to the concentration gradient of the perfluorocarbonvapor across the bubble film, the concentration of air surrounding thebubble, and the ratio of the bubble film permeability to air and toperfluorocarbon.

As discussed above, the Laplace pressure is inversely proportional tothe bubble radius; thus, as the bubble shrinks, the Laplace pressureincreases, increasing the rate of diffusion of gas out of the bubble andthe rate of bubble shrinkage, and in some cases leading to thecondensation and virtual disappearance of a gas in the bubble as thecombined Laplace and external pressures concentrate the osmotic agentuntil its partial pressure reaches the vapor pressure of liquid osmoticagent.

We have discovered that conventional microbubbles that contain anysingle gas will subsist in the blood for a length of time that dependsprimarily on the arterial pressure, the bubble diameter, the membranepermeability of the gas through the bubble's surface, the mechanicalstrength of the bubble's surface, the presence, absence, andconcentration of the gases that are ordinarily present in the blood orserum, and the surface tension present at the surface of the bubble(which is primarily dependent on the diameter of the bubble andsecondarily dependent on the identity and concentration of thesurfactants which form the bubble's surface). Each of these parametersare interrelated, and they interact in the bubble to determine thelength of time that the bubble will last in the blood.

The present invention includes the discovery that a single gas or acombination of gases can together act to stabilize the structure of themicrobubbles entraining or entrapping them. Essentially, the inventionutilizes a first gas or gases (a “primary modifier gas”) that optionallyis ordinarily present in normal blood and serum in combination with oneor more additional second gases (a “gas osmotic agent or agents” or a“secondary gas”) that act to regulate the osmotic pressure within thebubble. Through regulating the osmotic pressure of the bubble, the gasosmotic agent (defined herein as a single or mixture of chemicalentities) exerts pressure within the bubble, aiding in preventingdeflation. Optionally, the modifier gas may be a gas that is notordinarily present in blood or serum. However, the modifier gas must becapable of diluting and maintaining the gas osmotic agent or agents at apartial pressure below the vapor pressure of the gas osmotic agent oragents while the gases in blood or other surrounding liquid diffuse intothe bubble. In an aqueous medium, water vapor is not considered to beone of the “gases” in question. Similarly, when am microbubbles are in anonaqueous liquid medium, the vapor of that medium is not considered tobe one of the “gases.” We have discovered that by adding a gas osmoticagent that has, for example, a reduced membrane permeability through thebubble's surface or reduced solubility in the external continuous phaseliquid phase, the life of a bubble formed therewith will be radicallyincreased. This stabilizing influence can be understood more readilythrough a discussion of certain theoretical bubbles. First, we willconsider the effects of arterial pressure and surface tension on ahypothetical microbubble containing only air.

Initially, a hypothetical bubble containing only air is prepared. Forpurposes of discussion, this bubble will initially be considered to haveno Laplace pressure. Generally, when equilibrated at standardtemperature and pressure (STP), it will have a internal pressure of 760Torr of air and the surrounding fluid air concentration will also beequilibrated at 760 Torr (i.e., the fluid has an air tension of 760Torr). Such a bubble will neither shrink nor grow.

Next, when the above hypothetical bubble is introduced into the arterialsystem, the partial pressure of air (or air tension) in the blood (theair pressure at which the blood was saturated with air) will also beapproximately 760 Torr and there will be an arterial pressure (for thepurposes of the this discussion at 100 Torr). This total creates anexternal pressure on the bubble of 860 Torr, and causing the gases inthe bubble to be compressed until the internal pressure increases to 860Torr. There then arises a difference of 100 Torr between the airpressure inside the bubble and the air tension of the fluid surroundingthe bubble. This pressure differential causes air to diffuse out of thebubble, through its air-permeable surface membrane, causing the bubbleto shrink (i.e., lose air) as it strives to reach equilibrium. Thebubble shrinks until it disappears.

Next, consider the additional, and more realistic, effect on thehypothetical bubble of adding the surface tension of the bubble. Thesurface tension of the bubble leads to a Laplace pressure exerted on gasinside the bubble. The total pressure exerted on the gas inside thebubble is computed through adding the sum of the atmospheric pressure,the arterial pressure and the Laplace pressure. In a 3 μm bubble asurface tension of 10 dynes per centimeter is attainable with wellchosen surfactants. Thus, the Laplace pressure exerted on thehypothetical 3 μm bubble is approximately 100 Torr and, in addition, thearterial pressure of 100 Torr is also exerted on the bubble. Therefore,in our hypothetical bubble, the total external pressure applied to thegas inside the bubble is 960 Torr. The bubble will be compressed untilthe pressure of the air inside the bubble rises to 960 Torr.Accordingly, a concentration differential of 200 Torr arises between theair inside the bubble and the air dissolved in the blood. Therefore, thebubble will rapidly shrink and disappear even more rapidly than it didin the previous case, as it attempts to reach equilibrium.

The discovery of the present invention is illustrated by considering athird hypothetical microbubble containing air and a gas osmotic agent ora secondary gas. Assume that a theoretical bubble, initially having noarterial pressure and no Laplace pressure, is prepared having a totalpressure of 760 Torr, which is made up of air at a partial pressure of684 Torr and a perfluorocarbon (“PFC”) as a gas osmotic agent at apartial pressure of 76 Torr. Further, assume that the perfluorocarbon isselected to have one or more traits that make it capable of acting as anappropriate gas osmotic agent, such as limited bubble membranepermeability or limited solubility in the external liquid phase. Thereis an initial gas osmotic pressure differential between the 684 Torr ofair within the bubble and the 760 Torr of air tension outside the bubble(assuming STP) of 76 Torr. This 76 Torr initial pressure difference isthe initial gas osmotic pressure and will cause the bubble to expand.Air from outside of the bubble will diffuse into and inflate the bubble,driven by the osmotic pressure differential, similar to the way waterdiffuses into a dialysis bag containing a starch solution, and inflatesthe bag.

The maximum gas osmotic pressure this gas mixture can develop is relatedto the partial pressure of the PFC and the ratio of the permeability ofthe PFC to the permeability of the air in the surrounding fluid. Intheory, and as observed experimentally, the bubble will growindefinitely as the system attempts to reach osmotic equilibrium betweenthe concentration of air (equivalent to the partial pressure of air)within the bubble and the concentration of air surrounding the bubble(the air tension). When the hypothetical mixed gas bubble is exposed to100 Torr of arterial pressure where the blood has a dissolved airtension of 760 Torr, the total external pressure will equal 860 Torr(760 Torr atmospheric pressure and 100 Torr arterial pressure). Thebubble will compress under the arterial pressure, causing the internalpressure of the bubble to reach 860 Torr. The partial pressure of theair will increase to 774 Torr and the partial pressure of the PFC (thesecond gas) will increase to 86 Torr. The air will diffuse out of thebubble until it reaches osmotic equilibrium with the air dissolved inthe blood (i.e., 760 Torr) and the partial pressure of the PFC willincrease to 100 Torr. The partial pressure of the PFC will act tocounterbalance the pressure exerted due to the arterial pressure,halting shrinkage of the bubble, in each case, assuming that thepermeability of the bubble to the PFC is negligible.

When the surface tension or Laplace pressure component of 100 Torr isadded (as discussed above with the air bubble), a total of 200 Torradditional pressure is exerted on the gas in the bubble. Again, thebubble will compress until and the pressure inside the bubble increasesto 960 Torr (partial pressure of air 864 and partial pressure of PFC96). The air will diffuse from the bubble until it reaches 760 Torr (inequilibrium with the concentration of air dissolved in the blood) andthe partial pressure of the PFC will increase to 200 Torr, where, again,the gas osmotic pressure induced by the PFC will act to counterbalancethe pressure exerted by the Laplace pressure and the arterial pressure,again, assuming that the membrane permeability of the bubble to the PFCis negligible.

Similarly, if the partial pressure of air in the bubble is lower thanthe air tension in the surrounding liquid, the bubble will actually growuntil the PFC is sufficiently diluted by incoming air so that thepressure of air inside and the air tension outside of the bubble areidentical. Thus, bubbles can be stabilized effectively through the useof combinations of gases, since the correct combination of gases willresult in a gas osmotic pressure differential that can be harnessed tocounterbalance the effects of the Laplace pressure and the arterialpressure exerted on the a gas within the bubble in circulating blood.

Examples of particular uses of the microbubbles of the present inventioninclude perfusion imaging of the heart, the myocardial tissue, anddetermination of perfusion characteristics of the heart and its tissuesduring stress or exercise tests, or perfusion defects or changes due tomyocardial infarction. Similarly, myocardial tissue can be viewed afteroral or venous administration of drugs designed to increase the bloodflow to a tissue. Also, visualization of changes in myocardial tissuedue to or during various interventions, such as coronary tissue veingrafting, coronary angioplasty, or use of thrombolytic agents (TPA orstreptokinase) can also be enhanced. As these contrast agents can beadministered conveniently via a peripheral vein to enhance thevisualization of the entire circulatory system, they will also aid inthe diagnosis of general vascular pathologies and in the ability tomonitor the viability of placental tissue ultrasonically.

It should, however, be emphasized that these principles have applicationbeyond ultrasound imaging. Indeed, the present invention is sufficientlybroad to encompass the use of gas osmotic pressure to stabilize bubblesfor uses in any systems, including nonbiological applications.

In a preferred embodiment, the microbubbles of the present inventionhave a surfactant-based bubble membrane. However, the principles of theinvention can be applied to stabilize microbubbles of virtually anytype. Thus, mixed gases or vapors of the type described above canstabilize albumin based bubbles, polysaccharide based microbubbles,spray dried microsphere derived microbubbles, and the like. This resultis achieved through the entrapment, within the chosen microbubble, of acombination of gases, preferably a primary modifier gas or mixture ofgases that will dilute a gas osmotic agent to a partial pressure lessthan the gas osmotic agent's vapor pressure until the modifier gas willexchange with gases normally present in the external medium. The gasosmotic agent or agents are generally relatively hydrophobic andrelatively bubble membrane impermeable and also further possess theability to develop gas osmotic pressures greater than 50, 75, or 100Torr. In one preferred embodiment, the gas vapor pressure of the gasosmotic agent is preferably less than about 760 Torr at 37° C.,preferably less than about 750, 740, 730, 720, 710, or 700 Torr, and insome embodiments less than about 650, 600, 500, or 400 Torr. Inpreferred embodiments, the vapor pressure of the primary modifier gas isat least 660 Torr at 37° C. and the vapor pressure of the gas osmoticagent is at least 100 Torr at 37° C. For in vivo imaging mean bubblediameters between 1 and 10 μm are preferred, with 3 to 5 μm mostpreferred. The invention may in one embodiment also be described as amixture of a first gas or gases and a second gas or gases withingenerally spherical membranes to form microbubbles, where the first gasand the second gas are respectively present in a molar ratio of about1:100, 1:75, 1:50, 1:30, 1:20, or 1:10 to about 1000:1, 500:1, 250:1,100:1, 75:1 or 50:1, and where the first gas has a vapor pressure of atleast about (760−x) mm Hg at 37° C., where x is the vapor pressure ofthe second gas at 37° C., and where the vapor pressure of each of thefirst and second gases is greater than about 75 or 100 mm Hg at 37° C.

Microbubbles prepared in accordance with one preferred embodiment of theinvention may also possess an additional advantageous property. In onesuch embodiment, mixtures of nonosmotic gases with osmotic stabilizinggases (or gas osmotic agents) are used to stabilize the resultant bubblesize distribution during and immediately after production. Upongeneration of the bubbles, the higher Laplace pressure in smallerbubbles causes diffusion through the liquid phase to the lower Laplacepressure larger bubbles. This causes the mean size distribution toincrease above the capillary dimension limit of 5 microns over time.This is called disproportionation. When a mixture of a nonosmotic gas(e.g., air) is used with an osmotic vapor (e.g., C₆F₁₄) a slightreduction in volume of the smaller bubbles, due to air leaving thebubble, concentrates the osmotic gas and increases its osmotic pressurethus retarding further shrinkage while the larger bubbles increase involume slightly, diluting the osmotic gas and retarding further growth.

An additional advantage of using a mixture of an extremely blood solublegases (e.g., 87.5% by volume CO₂) and an osmotic gas mixture (e.g., 28%C₆F₁₄ vapor+72% air) is that, when injected, these bubbles rapidlyshrink due to the loss of CO₂ to the blood. The bubbles, upon injection,will experience an 87.5% volume decrease due to loss of CO₂. This lossof CO₂ corresponds to a halving of the bubble diameter. Accordingly, onecan prepare larger diameter bubbles (e.g., 9 μm), using simplifiedmechanical means, that will shrink to below 5 microns upon injection. Ingeneral, such bubbles will initially be prepared where the first gas ispresent in a ratio of at least 1:1 with respect to the second gas,preferably at least 3:2, 2:1, 3:1, 4:1, 5:1, or 10:1. Where themicrobubble membrane is more permeable to the first gas than to thesecond gas (e.g., the membrane has respective permeabilities to thegases in a ratio of at least about 2:1, 3:1, 4:1, 5:1, or 10:1,preferably even higher, e.g., 20:1, 40:1, or 100:1), the bubblesadvantageously shrink from their original first diameter to an averagesecond diameter of 75% or less of their original diameter quite rapidly(e.g., within one, two, four, or five minutes). Then, when at least onerelatively membrane-permeable gas is present in the aqueous mediumsurrounding the microbubble, the bubble is preferably stabilized at orabout the second diameter for at least about 1 minute, preferably for 2,3, 4, or 5 minutes. In one preferred embodiment, the bubbles maintain asize between about 5 or 6 μm and 1 μm for at least 1, 2, 3, 4, or 5minutes, stabilized by a gas osmotic pressure differential. The gastension in the external liquid is preferably at least about 700 mm Hg.Moreover, a relatively membrane impermeable gas is also in themicrobubble to create such an osmotic pressure differential.

I. Microbubble Construction

A. The Membrane-Forming Liquid Phase

The external, continuous liquid phase in which the bubble residestypically includes a surfactant or foaming agent. Surfactants suitablefor use in the present invention include any compound or compositionthat aids in the formation and maintenance of the bubble membrane byforming a layer at the interface between the phases. The foaming agentor surfactant may comprise a single compound or any combination ofcompounds, such as in the case of co-surfactants.

Examples of suitable surfactants or foaming agents include: blockcopolymers of polyoxypropylene polyoxyethylene, sugar esters, fattyalcohols, aliphatic amine oxides, hyaluronic acid aliphatic esters,hyaluronic acid aliphatic ester salts, dodecyl poly(ethyleneoxy)ethanol, nonylphenoxy poly(ethyleneoxy)ethanol, derivatized starches,hydroxy ethyl starch fatty acid esters, commercial food vegetablestarches, dextrans, dextran fatty acid esters, sorbitol, sorbitol fattyacid esters, gelatin, serum albumins, and combinations thereof.Particularly preferred sugar esters are those with a component having ahydrophilic-lipophilic balance (HLB) of less than 8 including mono-,di-, tri- or polyesterified sucrose, trehalose, dextrose and fructose toproduce stearate, behenate, palmitate, myristate, laurate and caproate(e.g. sucrose tristearate), as these sugar esters prolong microbubblestability. Other sugar esters contemplated for use in the presentinvention include those esterified with unsaturated fatty acids to formoleate, ricinoleate, linoleate, arachidate, palmitoleate andmyristoleate. The HLB is a number between 0 and 40 assigned toemulsifying agents and substances which are emulsified. The HLB isindicative of emulsification behavior and is related to the balancebetween the hydrophilic and lipophilic portions of the molecule (Rosen,M., (1989), Surfactants and Interfacial Phenomena, Second Edition, JohnWiley & Sons, New York, pp. 326-329).

In the present invention, preferred surfactants or foaming agents areselected from the group consisting of phospholipids, nonionicsurfactants, neutral or anionic surfactants, fluorinated surfactants,which can be neutral or anionic, and combinations of such emulsifying orfoaming agents.

The nonionic surfactants suitable for use in the present inventioninclude polyoxyethylene-polyoxypropylene copolymers. An example of suchclass of compounds is Pluronic, such as Pluronic F-68. Also contemplatedare polyoxyethylene fatty acids esters, such as polyoxyethylenestearates, polyoxyethylene fatty alcohol ethers, polyoxyethylatedsorbitan fatty acid esters, glycerol polyethylene glycol oxystearate,glycerol polyethylene glycol ricinoleate, ethoxylated soybean sterols,ethoxylated castor oils, and the hydrogenated derivatives thereof, andcholesterol. In addition, nonionic alkylglucosides such as Tweens®,Spans® and Brijs® having a component with an HLB less than 8 are alsowithin the scope of the present invention. The Spans include sorbitantetraoleate, sorbitan tetrastearate, sorbitan tristearate, sorbitantripalmitate, sorbitan trioleate, and sorbitan distearate. Tweensinclude polyoxyethylene sorbitan tristearate, polyoxyethylene sorbitantripalmitate, polyoxyethylene sorbitan trioleate. The Brij family isanother useful category of materials, which includes polyoxyethylene 10stearyl ether. Anionic surfactants, particularly fatty acids (or theirsalts) having 12 to 24 carbon atoms, may also be used. One example of asuitable anionic surfactant is oleic acid, or its salt, sodium oleate.

It will be appreciated that a wide range of surfactants can be used.Indeed, virtually any surfactant or foaming agent (including those stillto be developed) capable of facilitating formation of the microbubblescan be used in the present invention. The optimum surfactant or foamingagent or combination thereof for a given application can be determinedthrough empirical studies that do, not require undue experimentation.Consequently, one practicing the art of the present invention shouldchoose the surfactant or foaming agents or combination thereof basedupon such properties as biocompatibility or their non-Newtonianbehavior.

The blood persistence of a contrast agent is inversely proportional tothe Laplace pressure which is proportional to the surface tension of thebubble. Reduced surface tension, therefore, increases blood persistence.Surfactants that form ordered structures (multilaminar sheets and rods)in solution and produce non-Newtonian viscoelastic surface tensions areespecially useful. Such surfactants include many of the sugar basedsurfactants and protein or glycoprotein surfactants (including bovine,human, or other lung surfactants). One preferred type of such surfactanthas a sugar or other carbohydrate head group, and a hydrocarbon orfluorocarbon tail group. A large number of sugars are known that canfunction as head groups, including glucose, sucrose, mannose, lactose,fructose, dextrose, aldose, and the like. The tail group preferably hasfrom about 2 or 4 to 20 or 24 carbon atoms, and may be, for example, afatty acid group (branched or unbranched, saturated or unsaturated)covalently bound to the sugar through an ester bond. The surface tensionof bubbles produced with these surfactants greatly decreases as thesurface is compressed by shrinkage of the bubble (e.g., when the bubbleshrinks), and it is increased as the surface area of the bubble isincreased (e.g., when the bubble grows). This effect retardsdisproportionation, which leads to narrower size distribution and longerpersisting bubbles in the vial and in vivo. A preferred surfactantmixture that has the properties associated with non-Newtonianviscoelasticity includes a nonionic surfactant or other foamingsurfactant in combination with one of the non-Newtonian viscoelasticsurfactant such as one of the sugar esters (e.g. 2% Pluronic F-68 plus1% sucrose stearate). Often the ratio of the nonionic surfactant to thenon-Newtonian surfactant is from about 5:1 to about 1:5, with thesurfactants together (whether non-Newtonian or more conventional)comprising 0.5 to 8%, more preferably about 1 to 5% (w/v) of themicrobubble-forming liquid mixture.

The lowering of surface tension in small bubbles, counter to typicalLaplace pressure, allows the use of more efficient gas osmotic agentssuch as higher molecular weight perfluorocarbons as the gas osmoticagent. With conventional surfactants, the higher molecular weight PFCswill condense at the high bubble pressures. Without these efficientsurfactants higher boiling less membrane permeable PFCs, e.g. C₆F₁₄,would be extremely difficult.

One may also incorporate other agents within the aqueous phase. Suchagents may advantageously include conventional viscosity modifiers,buffers such as phosphate buffers or other conventional biocompatiblebuffers or pH adjusting agents such as acids or bases, osmotic agents(to provide isotonicity, hyperosmolarity, or hyposmolarity). Preferredsolutions have a pH of about 7 and are isotonic. However, when CO₂ isused as a first gas in a bubble designed to shrink rapidly to a firstsize, a basic pH can facilitate rapid shrinkage by removing CO₂ as itleaves the bubble, preventing a buildup of dissolved CO₂ in the aqueousphase.

B. The Gas Phase

A major aspect of the present invention is in the selection of the gasphase. As was discussed above, the invention relies on the use ofcombinations of gases to harness or cause differentials in partialpressures and to generate gas osmotic pressures, which stabilize thebubbles. The primary modifier gas is preferably air or a gas present inair. Air and/or fractions thereof are also present in normal blood andserum. Where the microbubbles are to be used in an environment differentfrom blood, the primary modifier gas is preferably selected from gasesnormally present in the external medium. Another criteria is the easewith which the primary modifier gas is diffused into or out of thebubbles. Typically, air and/or fractions thereof are also readilypermeable through conventional flexible or rigid bubble surfaces. Thesecriteria, in combination, allow for the rapid diffusion of the primarymodifier gas into or out of the bubbles, as required.

Modifier gases not present in the external medium can also be used.However, in this case the bubble will initially grow or shrink(depending on the relative permeability and concentrations of theexternal gases to the modifier) as the external gases replace theoriginal modifier gas. If, during this process, the gas osmotic agenthas not condensed, the bubble will remain stable.

The gas osmotic agent is preferably a gas that is less permeable throughthe bubble's surface than the modifier. It is also preferable that thegas osmotic agent is less soluble in blood and serum. Therefore, it willnow be understood that the gas osmotic agent can be a gas at room orbody temperature or it can ordinarily be a liquid at body temperature,so long as it has a sufficient partial or vapor pressure at thetemperature of use to provide the desired osmotic effect.

Accordingly, fluorocarbons or other compounds that are not gases at roomor body temperature can be used, provided that they have sufficientvapor pressure, preferably at least about 50 or 100 Torr at bodytemperature, or more preferably at least about 150 or 200 Torr. Itshould be noted that where the gas osmotic agent is a mixture of gases,the relevant measure of vapor pressure is the vapor pressure of themixture, not necessarily the vapor pressure of the individual componentsof the mixed gas osmotic agent.

It is also important that where a perfluorocarbon is used as the osmoticagent within a bubble, the particular perfluorocarbon does not condenseat the partial pressure present in the bubble and at body temperature.Depending on the relative concentrations of the primary modifier gas andthe gas osmotic agent, the primary modifier gas may rapidly leave thebubble causing it to shrink and concentrate the secondary gas osmoticagent. Such shrinking may occur until the gas osmotic pressure equalsthe external pressure on the bubble (maximum absolute arterial pressure)plus the Laplace pressure of the bubble minus the air tension, or airsaturation tension, of the blood (essentially one atmosphere). Thus thecondensing partial pressure of the resulting gas mixture at 37° C. mustbe above the equilibrium partial pressure, discussed above, of theosmotic agent.

Representative fluorocarbons meeting these criteria and in increasingability to stabilize microbubbles are as follows:

CCl₂F₂ 21 CF₄, CHClF₂<C₄F₁₀, N(C₂F₅)_(3l <C) ₅F₁₂<C₆F₁₄

Accordingly, it will be understood that PFC's with eight carbons atomsor fewer (37° C vapor pressures greater than 80 mm Hg) are preferred. Aswill also be understood, however, it is possible to construct largermolecules with increased volatility through the addition of heteroatomsand the like. Therefore, the determination of the optimal secondary gasosmotic agent or gases agents is not size limited, but, rather, is basedupon its ability to retain its vapor phase at body temperature and whileproviding a gas osmotic pressure equal to at least the sum of thearterial and Laplace pressures.

A listing of some compounds possessing suitable solubility and vaporpressure criteria is provided in Table I:

TABLE I perfluoro propanes, C₃F₈ perfluoro butanes, C₄F₁₀ perfluorocyclo butanes, C₄F₈ perfluoro pentanes, C₅F₁₂ perfluoro cyclo pentanes,C₅F₁₀ perfluoro methylcyclobutanes, C₅F₁₀ perfluoro hexanes, C₆F₁₄perfluoro cyclohexanes, C₆F₁₂ perfluoro methyl cyclopentanes, C₆F₁₂perfluoro dimethyl cyclobutanes, C₆F₁₂ perfluoro heptanes, C₇F₁₆perfluoro cycloheptanes, C₇F₁₄ perfluoro methyl cyclohexanes, C₇F₁₄perfluoro dimethyl cyclopentanes, C₇F₁₄ perfluoro trimethylcyclobutanes, C₇F₁₄ perfluoro triethylamines, N(C₂F₅₎ ₃

It will be appreciated that one of ordinary skill in the art can readilydetermine other compounds that would perform suitably in the presentinvention that do not meet both the solubility and vapor pressurecriteria, described above. Rather, it will be understood that certaincompounds can be considered outside the preferred range in eithersolubility or vapor pressure, if such compounds compensate for theaberration in the other category and provide a superior insolubility orlow vapor pressure.

It should also be noted that for medical uses the gases, both themodifier gas and the gas osmotic agent, should be biocompatible or notbe physiologically deleterious. Ultimately, the microbubbles containingthe gas phase will decay and the gas phase will be released into theblood either as a dissolved gas or as submicron droplets of thecondensed liquid. It will be understood that gases will primarily beremoved from the body through lung respiration or through a combinationof respiration and other metabolic pathways in the reticuloendothelialsystem.

Appropriate gas combinations of the primary modifier and secondary gasescan be ascertained empirically without undue experimentation. Suchempirical determinations are described in the Examples.

When an efficient surfactant, e.g., bovine lung surfactant, is employedto produce a large diameter bubble with a low surface tension, theLaplace pressure is very low. When perfluorooctylbromide (PFOB)saturated air is inside the bubble and the bubble is exposed to air or aliquid nearly saturated with air (e.g., equilibrated with air) the gasosmotic pressure is greater than the Laplace pressure and therefore thebubble grows. With smaller diameter bubbles the Laplace pressure ishigher and therefore the bubble shrinks and collapses. This shrinkage isat a reduced rate being driven by the difference between the Laplacepressure and the gas osmotic pressure. When small diameter bubbles arecreated by sonicating gas or gas vapor mixtures in a low surface tensionsurfactant solution, e.g., 2% pluronic F-68 plus 1% sucrose stearate,the time the bubbles persist in vitro, as observed by microscope, and invivo as observed by Doppler ultrasound imaging of a rabbit's kidney postintravenous injection, correlated with the above gas osmotic pressurecomparison.

In the rabbit kidney Doppler experiment (Example III), contrastenhancement was observed for up to 10 minutes with perfluorohexane/airmixtures in the bubbles compared with the instantaneous disappearance ofcontrast with pure air microbubbles. Thus, these perfluorochemicals arecapable of exerting gas osmotic pressures that nearly counterbalance theLaplace pressure and create functional ultrasound microbubble contrastagents.

A surprising discovery was that mixtures of PFCs, e.g., C₄F₁₀ (as acombination modifier gas and a gas osmotic agent) saturated with C₆F₁₄vapor (as the main gas osmotic agent), can stabilize the bubble forlonger times than either component alone. This is because C₄F₁₀ is a gasat body temperature (and, thus, can act as both a modifier gas and a gasosmotic agent) has a somewhat reduced membrane permeability and it isonly slightly soluble in C₆F₁₄ at body temperature. In this situationthe gas osmotic pressures of both agents are added together, leading toincreased bubble persistence over that of air/C₆F₁₄ only mixtures. It ispossible that the condensing point of the longer persisting highermolecular weight C₆F₁₄ component is increased, allowing a larger maximumgas osmotic pressure to be exerted. Other mixtures of PFCs will performsimilarly. Preferred mixtures of PFCs will have ratios of 1:10 to 10:1,and include such mixtures as perfluorobutane/perfluorohexane andperfluorobutane/perfluoropentane. These preferred fluorochemicals can bebranched or straight chain.

As was discussed above, we have also discovered that mixtures ofnonosmotic gases in combination with the gas osmotic agent act tostabilize the size distribution of the bubbles before and afterinjection. Upon generation of the bubbles, the higher Laplace pressuresin smaller bubbles causes diffusion through the liquid phase to thelower Laplace pressure larger bubbles. This causes the mean sizedistribution to increase above the capillary dimension limit of 5microns with time. This is called disproportionation.

However, when a mixture of a modifier gases (e.g., air or carbondioxide) are used with a gas osmotic agent (e.g., C₆F₁₄) a slightreduction in volume of the smaller bubbles, due to one of the modifiergases leaving the bubble, will concentrate the osmotic gas and increasesits osmotic pressure, thus, retarding further shrinkage. On the otherhand, the larger bubbles will increase in volume slightly, diluting theosmotic gas and also retarding further growth. An additional advantageof using a mixture of an extremely blood soluble gas (e.g., 75% through87.5% by volume CO₂) and an osmotic gas mixture (e.g. 28% C₆F₁₄ vaporand 72% air) is that when injected, these bubbles rapidly shrink due tothe loss of CO₂ to the blood. Carbon dioxide leaves particularly fastdue to a specific plasma enzyme that catalyzes its dissolution. An 87.5%volume decrease due to loss of CO₂ corresponds with a halving of thebubble diameter. Accordingly, larger diameter bubbles can be producedwhich will shrink to an appropriate size (i.e., 5 microns) uponinjection or exposure to a solution with a basic or alkaline pH.

Accordingly, we have discovered that through use of a gas that isrelatively hydrophobic and that has a relatively low membranepermeability, the rate of contrast particle decay can be reduced. Thus,through reducing the particle decay rate, the microbubbles' half livesare increased and contrast enhancement potential is extended.

II. Other Components.

It will be understood that other components can be included in themicrobubble formulations of the present invention. For example, osmoticagents, stabilizers, chelators, buffers, viscosity modulators, airsolubility modifiers, salts, and sugars can be added to fine tune themicrobubble suspensions for maximum life and contrast enhancementeffectiveness. Such considerations as sterility, isotonicity, andbiocompatibility may govern the use of such conventional additives toinjectable compositions. The use of such agents will be understood tothose of ordinary skill in the art and the specific quantities, ratios,and types of agents can be determined empirically without undueexperimentation.

III. Formation of the Microbubbles of the Present Invention.

There are a variety of methods to prepare microbubbles in accordancewith the present invention. Sonication is preferred for the formation ofmicrobubbles, i.e., through an ultrasound transmitting septum or bypenetrating a septum with an ultrasound probe including anultrasonically vibrating hypodermic needle. However, it will beappreciated that a variety of other techniques exist for bubbleformation. For example, gas injection techniques can be used, such asventuri gas injection.

Other methods for forming microbubbles include formation of particulatemicrospheres through the ultrasonication of albumin or other protein asdescribed in European Patent Application 0,359,246 by MolecularBiosystems, Inc.; the use of tensides and viscosity increasing agents asdescribed in U.S. Pat. No. 4,446,442; lipid coated, non-liposomal,microbubbles as is described in U.S. Pat. No. 4,684,479; liposomeshaving entrapped gases as is described in U.S. Pat. Nos. 5,088,499 and5,123,414; and the use of denatured albumin particulate microspheres asis described in U.S. Pat. No. 4,718,433. The disclosure of each of theforegoing patents and applications is hereby incorporated by reference.

Any of the above methods can be employed with similar success to entrainthe modifier gases and gas osmotic agents of the present invention.Moreover, it is expected that similar enhancement in longevity of thebubbles created will be observed through use of the invention.

Sonication can be accomplished in a number of ways. For example, a vialcontaining a surfactant solution and gas in the headspace of the vialcan be sonicated through a thin membrane. Preferably, the membrane isless than about 0.5 or 0.4 mm thick, and more preferably less than about0.3 or even 0.2 mm thick, i.e., thinner than the wavelength ofultrasound in the material, in order to provide acceptable transmissionand minimize membrane heating. The membrane can be made of materialssuch as rubber, Teflon, Mylar, urethane, aluminized film, or any othersonically transparent synthetic or natural polymer film or film formingmaterial. The sonication can be done by contacting or even depressingthe membrane with an ultrasonic probe or with a focused ultrasound“beam.” The ultrasonic probe can be disposable. In either event, theprobe can be placed against or inserted through the membrane and intothe liquid. Once the sonication is accomplished, the microbubblesolution can be withdrawn from and vial and delivered to the patient.

Sonication can also be done within a syringe with a low powerultrasonically vibrated aspirating assembly on the syringe, similar toan inkjet printer. Also, a syringe or vial may be placed in andsonicated within a low power ultrasonic bath that focuses its energy ata point within the container.

Other types of mechanical formation of microbubbles are alsocontemplated. For example, bubbles can be formed with a mechanical highshear valve (or double syringe needle) and two syringes, or an aspiratorassembly on a syringe. Even simple shaking may be used. The shrinkingbubble techniques described herein are particularly suitable formechanically formed bubbles, having lower energy input than sonicatedbubbles. Such bubbles will typically have a diameter much larger thanthe ultimately desired biocompatible imaging agent, but can be made toshrink to an appropriate size in accordance with the present invention.

In another method, microbubbles can be formed through the use of aliquid osmotic agent emulsion supersaturated with a modifier gas atelevated pressure introduced into in surfactant solution. Thisproduction method works similarly to the opening of soda pop, where thegas foams upon release of pressure forming the bubbles.

In another method, bubbles can be formed similar to the foaming ofshaving cream, where perfluorobutane, freon, or another like materialthat boils when pressure is released. However, in this method it isimperative that the emulsified liquid boils at sufficiently lowtemperatures or that it contain numerous bubble nucleation sites so asto prevent superheating and supersaturation of the aqueous phase. Thissupersaturation will lead to the generation of a small number of largebubbles on a limited number of nucleation sites rather than the desiredlarge number of small bubbles (one for each droplet).

In still another method, dry void-containing particles or otherstructures (such as hollow spheres or honeycombs) that rapidly dissolveor hydrate, preferably in an aqueous solution, e.g., albumin, microfinesugar crystals, hollow spray dried sugar, salts, hollow surfactantspheres, dried porous polymer spheres, dried porous hyaluronic acid, orsubstituted hyaluronic acid spheres, or even commercially availabledried lactose microspheres can be stabilized with a gas osmotic agent.

For example, a spray dried surfactant solution can be formulated byatomizing a surfactant solution into a heated gas such as air, carbondioxide, nitrogen, or the like to obtain dried 1-10 micron or largerhollow or porous spheres, which are packaged in a vial filled with anosmotic gas or a desired gas mixture as described herein. The gas willdiffuse into the voids of the spheres. Diffusion can be aided bypressure or vacuum cycling. When reconstituted with a sterile solutionthe spheres will rapidly dissolve and leave osmotic gas stabilizedbubbles in the vial. In addition, the inclusion of starch or dextrins, asugar polyester and/or an inflating agent such as methylene chloride,1,1,2-trichlorotrifluoroethane (Freon 113, EM Science, Gibbstown, N.J.)or perf luorohexane, will result in microbubbles with an increased invivo half-life. Particularly preferred starches for use in formation ofmicrobubbles include those with a molecular weight of greater than about500,000 daltons or a dextrose equivalency (DE) value of less than about12. The DE value is a quantitative measurement of the degree of starchpolymer hydrolysis. It is a measure of reducing power compared to adextrose standard of 100. The higher the DE value, the greater theextent of starch hydrolysis. Such preferred starches include food gradevegetable starches of the type commercially available in the foodindustry, including those sold under the trademarks N-LOK and CAPSULE byNational Starch and Chemical Co., (Bridgewater, N.J.); derivatizedstarches, such as hydroxyethyl starch (available under the trademarksHETASTARCH and HESPAN from du Pont Pharmaceuticals)(M-Hydroxyethylstarch, Ajinimoto, Tokyo, Japan). (Note that short chainstarches spray dry well and can be used to produce microbubbles, but arenot preferred because those with a molecular weight less than about500,000 do not stabilize the microbubbles. However, they can be used inthe present invention in applications in which additional stabilizationis not required.) In the alternative, a lyophilized cake of surfactantand bulking reagents produced with a fine pore structure can be placedin a vial with a sterile solution and a head spaced with an osmotic gasmixture. The solution can be frozen rapidly to produce a fine icecrystal structure and, therefore, upon lyophilization produces finepores (voids where the ice crystals were removed).

Alternatively, any dissolvable or soluble void-forming structures may beused. In this embodiment, where the void-forming material is not madefrom or does not contain surfactant, both surfactant and liquid aresupplied into the container with the structures and the desired gas orgases. Upon reconstitution these voids trap the osmotic gas and, withthe dissolution of the solid cake, form microbubbles with the gas orgases in them.

It will be appreciated that kits can be prepared for use in making themicrobubble preparations of the present invention. These kits caninclude a container enclosing the gas or gases described above forforming the microbubbles, the liquid, and the surfactant. The containercan contain all of the sterile dry components, and the gas, in onechamber, with the sterile aqueous liquid in a second chamber of the samecontainer. Suitable two-chamber vial containers are available, forexample, under the trademarks WHEATON RS177FLW or S-1702FL from WheatonGlass Co., (Millville, N.J.). Such a container is illustrated in FIGS.1-4. Referring to FIGS. 1 and 2, the illustrated Wheaton container 5 hasan upper chamber 20 which can contain an aqueous solution 25, and alower chamber 30 which can contain the dried ingredients 35 and adesired gas. A stopper 10 is provided separating the top chamber fromthe environment, and a seal 15 separates the upper chamber 20 from thelower chamber 30 containing spray dried (powdered) hollow microsphere 35and the gas osmotic agent. Depressing the stopper 10 pressurizes therelatively incompressible liquid, which pushes the seal 15 downward intothe lower chamber 30. This releases aqueous solution 25 into lowerchamber 30, resulting in the dissolution of powder 35 to form stabilizedmicrobubbles 45 containing the entrapped gas osmotic agent. Excess gasosmotic agent 40 is released from the lower chamber 30 into Ad the upperchamber 20. This arrangement is convenient for the user and has theadded unexpected advantage of sealing the small quantity ofwater-impermeable gas osmotic agent in the lower chamber by covering theinterchamber seal with a thick (0.5 to 1.25 inch) layer of aqueoussolution and the advantage that the aqueous solution can be introducedinto the lower chamber without raising the pressure in the powderchamber by more than about 10%. Thus, there is no need for a pressurevent. (In contrast, conventional reconstitution of a solute in a singlechamber vial with a needle and syringe without a vent can result in theproduction of considerable intrachamber pressure which could collapsethe microbubbles.) The formation of microbubbles by this method isdescribed in Example XIII.

Alternatively, an inverted two-chamber vial may be used for microbubblepreparation.. Referring to FIGS. 3 and 4, the same vial is used asdescribed hereinabove, except that the stopper 50 is elongated such thatit dislodges inner seal 15 when depressed. In this microbubblepreparation method, the spray dried hollow microspheres 35 and the gasosmotic agent 40 are contained in the upper chamber 20. The aqueoussolution 25 and gas osmotic agent 40 are contained within lower chamber30. When stopper 50 is depressed, it dislodges seal 15 allowing thespray dried hollow microspheres to mix with the aqueous solution 25 inthe presence of gas osmotic agent 40. One advantage asociated with thismethod of microbubble formation is that the aqueous phase can beinstilled first and sterilized via autoclaving or other means, followedby instillation of the spray dried microspheres. This will preventpotential microbial growth in the aqueous phase prior to sterilization.

Although one particular dual chamber container has been illustrated,other suitable devices are known and are commercially available. Forexample, a two compartment glass syringe such as the B-D HYPAKLiquid/Dry 5+5 ml Dual Chamber prefilled syringe system (BectonDickinson, Franklin Lakes, N.J.; described in U.S. Pat. No. 4,613,326)can advantageously be used to reconstitute the spray dried powder. Theadvantages of this system-include:

1. Convenience of use;

2. The aqueous-insoluble gas osmotic agent is sealed in by a chamber ofaqueous solution on one side and an extremely small area of elastomersealing the needle on the other side; and

3. a filtration needle such as Monoject #305 (Sherwood Medical, St.Louis, Mo.) can be fitted onto the syringe at the time of manufacture toensure that no undissolved solids are injected.

The use of the two chamber syringe to form microbubbles is described inExample XIV.

It can be appreciated by one of ordinary skill in the art that othertwo-chamber reconstitution systems capable of combining the spray driedpowder with the aqueous solution in a sterile manner are also within thescope of the present invention. In such systems, it is particularlyadvantageous if the aqueous phase can be interposed between thewater-insoluble osmotic gas and the environment, to increase shelf alife of the product.

Alternatively, the container can contain the void forming material andthe gas or gases, and the surfactant and liquid can be added to form themicrobubbles. In one embodiment, the surfactant can be present with theother materials in the container, so that only the liquid needs to beadded in order to form the microbubbles. Where a material necessary forforming the microbubbles is not already present in the container, it canbe packaged with the other components of the kit, preferably in a formor container adapted to facilitate ready combination with the othercomponents of the kit.

The container used in the kit may be of the type described elsewhereherein. In one embodiment, the container is a conventional septum-sealedvial. In another, it has a means for directing or permitting applicationof sufficient bubble forming energy into the contents of the container.This means can comprise, for example, the thin web or sheet describedpreviously.

Various embodiments of the present invention provide surprisingadvantages. The spray dried starch formulations give prolonged in-vialstability of the microbubbles, particularly when the molecular weight ofthe starch is over about 500,000. Use of fatty acid esters of sugarscontaining a component with an HLB of less than 8 provides greatlyincreased in vivo and in-vial stability. Fatty acid esters of sugarssuch as sucrose monosterate, as well as block copolymers such asPluronic F-68 (with an HLB over 12) allow the powder to form bubbles atthe instant they are rehydrated. Spray dried formulations with astructural agent such as a starch, starch derivative, or dextrin providea significantly lower total dose of surfactant than comparable sonicatedformulations. The use of two-chamber vials with water providing anadditional seal for the fluorocarbon gas provide increased shelf life,and greater use convenience. Spray dried formulations with a structuralagent (such as a starch or dextrin) and a sugar polyester providemicrobubbles with greatly increased in vivo half lives.

The inclusion of an inflating agent in the solution to be spray-driedresults in a greater ultrasound signal per gram of spray-dried powder byforming a greater number of hollow microspheres. The inflating agentnucleates steam bubble formulation within the atomized droplets of thesolution entering the spray dryer as these droplets mix with the hot airstream within the dryer. Suitable inflating agents are those thatsupersaturate the solution within the atomized droplets with gas orvapor, at the elevated temperature of the drying droplets (approximately100° C.).

Suitable agents include:

1. Dissolved low-boiling (below 100° C.) solvents with limitedmiscibility with aqueous solutions, such as methylene chloride, acetoneand carbon disulfide used to saturate the solution at room temperature.

2. A gas, e.g. CO₂ or N₂, used to saturate the solution at roomtemperature and elevated pressure (e.g. 3 bar) The droplets are thensupersaturated with the gas at 1 atmosphere and 100° C.

3. Emulsions of immiscible low-boiling (below 100° C.) liquids such asFreon 113, perfluoropentane, perfluorohexane, perfluorobutane, pentane,butane, FC-11, FC-11B1, FC-11B2, FC-12B2, FC-21, FC-21B1, FC-21B2,FC-31B1, FC-113A, FC-122, FC-123, FC-132, FC-133, FC-141, FC-141B,FC-142, FC-151, FC-152, FC-1112, FC-1121 and FC-1131.

The inflating agent is substantially evaporated during the spray dryingprocess and thus is not present in the final spray-dried powder in morethan trace quantities.

The inclusion of the surfactants and wetting agents into the shell ofthe microsphere allows the use of a lower surfactant concentration. Asthe microsphere shell is dissolving, it temporarily surrounds themicrobubble formed in its interior with a layer of aqueous phase that issaturated with the surfactants, enhancing their deposition on themicrobubble's surface. Thus, spray-dried surfactant containingmicrospheres require only locally high concentrations of surfactant, andobviate the need for a high surfactant concentration in the entireaqueous phase.

Any of the microbubble preparations of the present invention may beadministered to a vertebrate, such as a bird or a mammal, as a contrastagent for ultrasonically imaging portions of the vertebrate. Preferably,the vertebrate is a human, and the portion that is imaged is thevasculature of the vertebrate. In this embodiment, a small quantity ofmicrobubbles (e.g., 0.1 ml/Kg [2 mg/Kg spray-dried powder] based on thebody weight of the vertebrate) is introduced intravascularly into theanimal. Other quantities of microbubbles, such as from about 0.005 ml/Kgto about 1.0 ml/Kg, can also be used. Imaging of the heart, arteries,veins, and organs rich in blood, such as liver, lungs, and kidneys canbe ultrasonically imaged with this technique. The foregoing descriptionwill be more fully understood with reference to the following Examples.Such Examples, are, however, exemplary of preferred methods ofpracticing the present invention and are not limiting of the scope ofthe invention or the claims appended hereto.

EXAMPLE I

Preparation of microbubbles through sonication

Microbubbles with an average number weighted size of 5 microns wereprepared by sonication of an isotonic aqueous phase containing 2%Pluronic F-68 and 1% sucrose stearate as surfactants, air as a modifiergas and perfluorohexane as the gas osmotic agent.

In this experiment, 1.3 ml of a sterile water solution containing 0.9%NaCl, 2% Pluronic F-68 and 1% sucrose stearate was added to a 2.0 mlvial. The vial had a remaining head space of 0.7 ml initially containingair. Air saturated with perfluorohexane vapor (220 torr ofperfluorohexane with 540 torr of air) at 25 degrees C. was used to flushthe headspace of the vial. The vial was sealed with a thin 0.22 mmpolytetrafluoroethylene (PTFE) septum. The vial was turned horizontally,and a ⅛″ (3 mm) sonication probe attached to a 50 watt sonicator modelVC50, available from Sonics & Materials was pressed gently against theseptum. In this position, the septum separates the probe from thesolution. Power was then applied to the probe and the solution wassonicated for 15 seconds, forming a white solution of finely dividedmicrobubbles, having an average number weighted size of 5 microns asmeasured by Horiba LA-700 laser light scattering particle analyzer.

EXAMPLE II

Measurement of in-vitro size of microbubbles

The in-vitro size of the microbubbles prepared in Example I was measuredby laser light scattering. Studies of bubbles were conducted where themicrobubbles were diluted into a 4% dextrose water solution (1:50)circulating through a Horiba LA-700 laser light scattering analyzer. Theaverage microbubbles size was 5 microns and doubled in size in 25minutes.

Interestingly, microbubbles prepared through the same method in ExampleI without the use of a gas osmotic agent (substituting air for the perfluorohexane/air mixture) had an average size of 11 microns and gave onlybackground readings on the particle analyzer at 10 seconds.

EXAMPLE III

Measurement of in-vivo lifetime of microbubbles

The lifetimes of microbubbles prepared in accordance with Example I weremeasured in rabbits through injecting 0.2 ml of freshly formedmicrobubbles into the marginal ear vein of a rabbit that was underobservation with a Accuson 128XP/5 ultrasound imaging instrument with a5 megahertz transducer. Several tests were conducted, during whichimages of the heart, inferior vena cava/aorta, and kidney were obtainedwhile measuring the time and extent of the observable contrastenhancement. The results are presented in the following Table II:

TABLE II TIME TO MINIMUM TIME MAX. USABLE TIME TO NO ORGAN DOSEINTENSITY INTENSITY ENHANCEMENT Heart 0.1 ml/Kg 7-10 sec. 8-10 min. 25min. IVC/Aorta 0.1 ml/Kg 7-10 sec. 8-10 min. 25 min. Kidney 0.1 ml/Kg7-10 sec. 8-10 min. 25 min.

In Table III, a comparison of microbubbles prepared in an identicalfashion without the use of an osmotic gas is presented (only air wasused). Note that sporadic reflections were observed only in the rightheart ventricle during the injection but disappeared immediately postdosing.

TABLE III TIME TO TIME TO MINIMUM MAXIMUM USABLE TIME TO NO ORGAN DOSEINTENSITY INTENSITY ENHANCEMENT Heart 0.1 ml/Kg 0 0 0 IVC/Aorta 0.1ml/Kg 0 0 0 Kidney 0.1 ml/Kg 0 0 0

The use of an osmotic agent dramatically increased the length of timefor which microbubbles are visible.

EXAMPLE IV

Preparation of mixed osmotically stabilized microbubbles

Microbubbles with an average number weighted size of 5 microns wereprepared by sonication of an isotonic aqueous phase containing 2%Pluronic F-68 and 1 % sucrose stearate as surfactants and mixtures ofperfluorohexane and perfluorobutane as the gas osmotic agents.

In this experiment, 1.3 ml of a sterile water solution containing 0.9%NaCl and 2% Pluronic F-68 was added to a 2.0 ml vial. The vial had aremaining head space of 0.7 ml, initially containing air. An osmotic gasmixture of perfluorohexane, 540 Torr and perfluorobutane at 220 Torr wasused to flush the headspace before sealing with a thin 0.22 mm PTFEseptum. The vial was sonicated as in Example I, forming a white solutionof finely divided microbubbles, having an average particle size of 5microns as measured by a Horiba LA-700 laser light scattering particleanalyzer. This procedure was repeated twice more, once with pureperfluorobutane and then with a 540 Torr air +220 Torr perfluorohexanemixture. Vascular persistence of all three preparations was determinedby ultrasound imaging of a rabbit post I.V. injection and are listedbelow

1.5 minutes perfluorobutane   2 minutes perfluorohexane + air   3minutes perfluorobutane + perfluorohexane

The mixture of perfluorocarbons persisted longer than either agentalone.

EXAMPLE V

Preparation of gas osmotically stabilized microbubbles from solublespray dried spheres

Gas osmotically stabilized microbubbles were prepared by dissolvinghollow spray dried lactose spheres, filled with an air perfluorohexanevapor mixture, in a surfactant solution.

Spray dried spheres of lactose with a mean diameter of approximately 100micron and containing numerous 10 to 50 micron cavities, was obtainedfrom DMV International under the trade name of PHARMATOSE DCL-11. Ninetymilligrams of the lactose spheres was placed in a 2.0 ml vial. Theporous spheres were filled with a mixture of 220 Torr perfluorohexaneand 540 Torr air by cycling the gas pressure in the vial between oneatmosphere and ½ atmosphere a total of 12 times over 5 minutes. Asurfactant solution containing 0.9%- sodium chloride, 2% Pluronic-F68and 1% sucrose stearate was warmed to approximately 45° C., to speed thedissolution of the lactose, before injecting 1.5 ml of the warmedsolution into the vial. The vial was then gently agitated by inversionfor approximately 30 seconds to dissolve the lactose before injectingthe microbubbles thus prepared into the Horiba LA-700 particle analyzer.A 7.7 micron volume weighted median diameter was measured approximatelyone minute after dissolution. The diameter of these microbubblesremained nearly constant, changing to a median diameter of 7.1 micronsin 10 minutes. When the experiment was repeated with air filled lactose,the particle analyzer gave only background readings one minute afterdissolution, thus demonstrating that gas osmotically stabilizedmicrobubbles can be produced by the dissolution of gas-filledcavity-containing structures.

EXAMPLE VI

Preparation of larger bubbles that shrink to a desired size

Microbubbles with an average volume weighted size of 20 micronsshrinking to 2 microns were prepared by sonication of an isotonicaqueous phase containing 2% Pluronic F-68 as the surfactant, CO₂ as adiluent gas and perfluorohexane as the gas osmotic agent.

In this experiment, 1.3 ml of a sterile water solution containing 0.9%NaCl, 2% Pluronic F-68 and 1% sucrose stearate was added to a 2.0 mlvial. The vial had a remaining head space of 0.7 ml initially containingair. A mixture of air saturated with perfluorohexane at 25 degrees C.diluted by a factor of 10 with CO₂ (684 Torr CO₂+54 Torr air+22 Torrperfluorohexane) was used to flush the head space. The vial was sealedwith a thin 0.22 mm PTFE septum. The vial was sonicated as in Example I,forming a white solution of finely divided microbubbles, having anaverage particle size of 28 microns as measured by Horiba LA-700 laserlight scattering analyzer. In the 4% dextrose+0.25mM NaOH solution ofthe Horiba, the average bubble diameter rapidly shrank in 2 to 4 minutesfrom 28 microns to 5 to 7 microns, and then remained relatively constant, reaching 2.6 micron after 27 minutes. This is because the CO₂ leavesthe microbubbles by dissolving into the water phase.

EXAMPLE VII

Perfluoroheptane stabilized microbubble in vitro experiment

Microbubbles were prepared as in Example I above employingperfluoroheptane saturated air (75 torr plus 685 torr air) and weremeasured as in Example II above. The average number weighted diameter ofthese microbubbles was 7.6 micron, one minute after circulation, and 2.2microns after 8 minutes of circulation. This persistence, compared tothe near immediate disappearance of microbubbles containing only air,demonstrates the gas osmotic stabilization of perfluoroheptane.

EXAMPLE VIII

Perfluorotripropyl amine stabilized microbubble in vivo experiment

Microbubbles were prepared as in Example I above, employingperfluorotripropyl amine saturated air and were assessed as in ExampleIII above. The usable vascular persistence of these microbubbles wasfound to be 2.5 minutes, thus demonstrating the gas osmoticstabilization of perfluorotripropyl amine.

EXAMPLE IX

Effect of a non Newtonian viscoelastic surfactant - sucrose stearate

Microbubbles were prepared as in Example I above employing 0.9% NaCl, 2%Pluronic F-68 and 2% sucrose stearate as the surfactant and withperfluoropropane saturated air and perfluorohexane saturated air in theheadspace. These two preparations were repeated with the same surfactantsolution minus sucrose stearate. All four microbubble preparations wereassessed as in Example III above. The usable vascular persistence ofthese microbubbles are listed below:

2% Pluronic F-68 +2% sucrose stearate persistence

2 minutes perfluoropropane

4 minutes perfluorohexane

2% Pluronic F-68 only persistence

2 minutes perfluoropropane

1 minute perfluorohexane

As seen above, the reduced surface tension made possible by thenon-Newtonian viscoelastic properties of sucrose stearate prevented theless volatile perfluorohexane from condensing, allowing perfluorohexanemicrobubbles of longer persistence to be produced.

EXAMPLE X

Spray drying of starch-containing emulsion

One liter of each of the following solutions was prepared with water forinjection: Solution A containing 4.0% w/v N-Lok vegetable starch(National Starch and Chemical Co., Bridgewater, N.J.) and 1.9% w/vsodium chloride (Mallinckrodt, St. Louis, Mo.) and Solution B containing2.0% Superonic F-68 (Serva, Heidelberg, Germany) and 2.0% w/v RyotoSucrose Stearate S-1670 (Mitsubishi-Kasei Food Corp., Tokyo, Japan).Solution B was added to a high shear mixer and cooled in an ice bath. Acoarse suspension of 40 ml 1,1,2-trichlorotrifluoroethane (Freon 113; EMScience, Gibbstown, N.J.) was made in the 1 liter of solution B. Thissuspension was emulsified using a Microfluidizer (MicrofluidicsCorporation, Newton, Mass.; model M-110F) at 10,000 psi, 5° C. for 5passes. The resulting emulsion was added to solution A to produce thefollowing formula for spray drying:

2.0% w/v N-Lok

1.0% w/v Superonic F-68

1.0% w/v Sucrose Stearate S-1670

0.95% w/v sodium chloride

2.0% v/v 1,1,2-trichlorotrifluoroethane

This emulsion was then spray dried in a Niro Atomizer Portable SprayDryer equipped with a two fluid atomizer (Niro Atomizer, Copenhagen,Denmark) employing the following settings:

hot air flow rate=39.5 CFM

inlet air temp.=235° C.

outlet air temp.=10° C.

atomizer air flow=110 liters/hr

emulsion feed rate=1 liter/hr

The dry, hollow spherical product had a diameter between about 1 μM andabout 15 μM and was collected at the cyclone separator as is standardfor this dryer. Aliquots of powder (250 mg) were weighed into 10 mltubing vials, sparged with perfluorohexane-saturated nitrogen at 13° C.and sealed. The nitrogen was saturated with perfluorohexane by passingit through three perfluorohexane filled gas washing bottles immersed ina 13° C. water bath.

The vials were reconstituted with 5 ml water for injection afterinserting an 18-gauge needle as a vent to relieve pressure as the waterwas injected. One ml of the resulting microbubble suspension wasinjected intravenously into an approximately 3 kg rabbit instrumented tomonitor the Doppler ultrasound signal of its carotid artery. A 10 MHzflow cuff (Triton Technology Inc., San Diego, Calif.; model ES-10-20)connected to a System 6 Doppler flow module (Triton Technology Inc.) fedthe RF doppler signal to a LeCroy 9410 oscilloscope (LeCroy, ChestnutRidge, N.Y.). The root mean square (RMS) voltage of the signal computedby the oscilloscope was transferred to a computer and the resultantcurve fitted to obtain peak echogenic signal intensity and half-life ofthe microbubbles in blood. Signals before contrast were less than 0.1volts RMS. A peak signal of 1.6 volts RMS was observed with a half lifeof 52.4 seconds, thus demonstrating the ability of spray dried powdersto produce intravascular echogenic microbubbles that have a prolongedhalf-life in vivo.

EXAMPLE XI

Production of microbubbles containing hydroxyethyl starch

An emulsion for spray drying was prepared as in Example X to give afinal composition of:

2.0% w/v m-HES hydroxyethylstarch (Ajinimoto, Tokyo, Japan)

2.0% w/v sodium chloride (Mallinckrodt)

1.74% sodium phosphate, dibasic (Mallinckrodt)

0.26% w/v sodium phosphate, monobasic (Mallinckrodt)

1.7% w/v Superonic F-68 (Serva)

0.2% w/v Ryoto Sucrose Stearate S-1670 (Mitsubishi-Kasei Food Corp.)

0.1% w/v Ryoto Sucrose Stearate S-570 (Mitsubishi-Kasei Food Corp.)

4.0% w/v 1,1,2-trichlorotrifluoroethane (Freon 113; EM Science,Gibbstown, N.J.)

This emulsion was spray dried as in Example X using the followingparameters:

hot air flow rate=39.5 CFM

inlet air temperature=220° C.

outlet air temperature=105° C.

atomizer air flow=110 liter/hr

emulsion feed rate=1 liter/hr

Sample vials were prepared with 400 mg spray dried ERIE powder andsparged as in Example X. After reconstitution with 5 ml water forinjection and intravenous administration of 1.0 ml to a rabbit as inExample X, a peak signal of 2.4 volts RMS was observed with a half lifeof 70.9 seconds. This demonstrates the ability of higher molecularweight and derivatized injectable grade starches and sugar polyesters toproduce higher in vivo signals which persist longer as compared tostandard vegetable starch (Example X). This also demonstrates the lowersurfactant concentration required by an optimal spray dried formula andthe use of a spray drying inflating agent such as Freon 113.

EXAMPLE XII

Stability of starch-containing microbubbles

The spray dried powder described in Example X was reconstituted asdescribed. For comparison, the sonicated microbubbles from Example Iwere prepared and both were examined by light microscopy within 2minutes of preparation and again at 10 minutes after preparation. Thesonicated product was found to have a wider initial bubble diameterdistribution (between about 1 and about 30 μM) than the reconstitutedspray dried powder (between about 3 and about 15 μM). After 10 minutes,the vials were agitated, resampled and again observed by lightmicroscopy. The reconstituted spray dried product was essentiallyunchanged, while the sonicated microbubbles had grown bydisproportionation until nearly all of the observable microbubbles weremore than 10 μM in diameter. This experiment demonstrates the extendedin-vial stability of microbubbles produced by spray drying and theability of starches to increase in vitro stability.

EXAMPLE XIII

Microbubble formation using two chamber vial

800 mg spray dried powder from Example XII were weighed into the lowerchamber of a 20 ml Wheaton RS-177FLW two chamber vial (FIG. 1). The vialwas flushed with perfluorohexane-saturated nitrogen at 13° C. beforeinserting the interchamber seal. The upper chamber was filled with 10 mlsterile water for injection. The upper chamber stopper was inserted soas to eliminate all air bubbles in the upper chamber. Upon depression ofthe upper stopper, the interchamber seal was forced into the lowerchamber, allowing the water to flow into the lower chamber andreconstitute the powder (FIG. 2). Numerous stable microbubbles wereformed as demonstrated by light microscopy. This procedure demonstratesthe convenience of this form of packaging and the elimination of theneed to provide a vent to eliminate pressure buildup when the aqueousphase is added to the powder.

EXAMPLE XIV

Microbubble formation using two chamber syringe

One hundred mg of the spray dried powder from Example XI was weighedinto a 5 ml+5 ml HYPAK Liquid/Dry dual chamber syringe (BectonDickinson, Franklin Lakes, N.J.) and shaken into the powder (needle end)chamber. The interchamber seal was then positioned just above the bypasschannel. A 5 μM filter-containing needle was then fitted on the syringe.The powder-containing chamber was then filled with the gas osmotic agentby placing the assembly in a vacuum chamber, evacuating and refillingthe chamber with the gas osmotic agent, perfluorohexane-saturatednitrogen at 13° C. The filter needle allows the evacuation and refillingof the atmosphere in the powder-containing chamber. A sealing needlecover was then placed on the needle. The liquid chamber was then filledwith 4 ml water for injection and the plunger was seated using atemporary vent (wire inserted between the glass syringe barrel and theplunger so as to eliminate all air bubbles.

To reconstitute, the needle sealing cover was removed to eliminatepressure buildup in the powder chamber. The plunger was then depressed,forcing the interchamber seal to the bypass position which allowed thewater to flow around the interchamber seal into the powder-containingchamber. The plunger motion was stopped when all the water was in thepowder chamber. The syringe was agitated to dissolve the powder. Excessgas and any large bubbles were expelled by holding the syringe,,needleend up, and further depressing the plunger. The solution containingnumerous stabilized microbubbles (as observed by light microscopy) wasthen expelled from the syringe by depressing the plunger to its limit.

The foregoing description details certain preferred embodiments of thepresent invention and describes the best mode contemplated. It will beappreciated, however, that no matter how detailed the foregoing appearsin text, the invention can be practiced in many ways and the inventionshould be construed in accordance with the appended Claims and anyequivalents thereof.

What is claimed is:
 1. A method of imaging an object, a body part or abody cavity comprising the steps of: introducing into said object, bodypart or body cavity a microbubble preparation comprising an aqueousmedium having dispersed therein a plurality of osmotically stabilizedmicrobubbles, said microbubbles comprising a generally sphericalmicrobubble membrane containing at least one modifier gas and at leastone gas osmotic agent, wherein said modifier gas and said gas osmoticagent are present in a molar ratio from about 1:100 to about 1,000:1,wherein said ratio is effective to stabilize said microbubblepreparation, with the proviso that said modifier gas is not water vapor,wherein said gas osmotic agent is selected from the group consisting ofperfluoropentane, perfluorocyclopentane, perfluoromethylcyclopentane,perfluorodimethylcyclobutane, perfluorohexane, perfluorocyclohexane,perfluoroheptane, perfluorocycloheptane, perfluoromethylcyclohexane,perfluorodimethylcyclopentane, perfluorotrimethylcyclobutane,perfluorotriethylamine and combinations thereof; and imaging at least aportion of said object, body part or body cavity by ultrasound ormagnetic resonance.
 2. The method of claim 1 wherein said modifier gasis a fluorocabon gas.
 3. The method of claim 2 wherein said fluorocarbonmodifier gas is selected from the group comprising perfluoropropane,perfluorobutane, perfluorocyclobutaxe, perfluoromethylcyclobutane,perfluoropentane and perfluorocyclopentane.
 4. The method of claim 1wherein said modifier gas is a nonfluorocarbon gas.
 5. The method ofclaim 4 wherein said nonfluorocarbon modifier gas is selected from thegroup consisting of nitrogen, oxygen, carbon dioxide and mixturesthereof.
 6. The method of claim 1, wherein said gas osmotic agent has awater solubility of not more than about 0.5 mM at 25° C. and oneatmosphere.
 7. The method of claim 1 wherein the microbubble membranecomprises a surfactant.
 8. The method of claim 7 wherein said surfactantis selected from the group consisting of nonionic surfactants, neutralsurfactants, anionic surfactants, neutral fluorinated surfactants,anionic fluorinated surfactants and combinations thereof.
 9. The methodof claim 7 wherein said surfactant is a non-Newtonian surfactant. 10.The method of claim 7 wherein said surfactant is selected from the groupconsisting of polyoxypropylene polyoxyethylene copolymers, sugar esters,fatty alcohols, aliphatic amine oxides, hyaluronic acid aliphaticesters, hyaluronic acid aliphatic ester salts, dodecylpoly(ethyleneoxy)ethanol, nonylphenoxy poly(ethyleneoxy)ethanol, hydroxyethyl starch, hydroxy ethyl starch fatty acid esters, dextran fatty acidesters, sorbitol, sorbitol fatty acid esters, gelatin, serum albumins,phospholipids, polyoxyethylene fat acids esters polyoxyethylene fattyalcohol ethers, polyoxyethylated sorbitan fatty acid esters, glycerolpolyethylene glycol oxystearate, glycerol polyethylene glycolricinoleate, ethoxylated soybean sterols, ethoxylated castor oil,cholesterol, oleic acid, sodium oleate and combinations thereof.
 11. Themethod of claim 1 wherein the microbubble membrane comprises a liposome.12. The method of claim 1 wherein the microbubble membrane comprises aproteinaceous material.
 13. The method of claim 12 wherein saidproteinaceous material is albumin.
 14. The method of claim 1 whereinsaid gas osmotic agent is perfluoropentane.
 15. The method of claim 14wherein said modifier gas is a fluorocabon modifier gas.
 16. The methodof claim 14 wherein said modifier gas is a nonfluorocarbon modifier gas.17. The method of claim 16 wherein said nonfluorocarbon modifier gas isselected from the group consisting of nitrogen, oxygen, carbon dioxideand mixtures thereof.
 18. The method of claim 16 wherein saidnonfluorocarbon modifier gas is air.
 19. The method of claim 14 whereinthe microbubble membrane comprises a surfactant.
 20. The method of claim19 wherein said surfactant is selected from the group consisting ofnonionic surfactants, neutral surfactants, anionic surfactants, neutralfluorinated surfactants, anionic fluorinated surfactants andcombinations thereof.
 21. The method of claim 19 wherein said surfactantis a fluorinated surfactant.
 22. The method of claim 14 wherein themicrobubble membrane comprises a proteinaceous material.
 23. The methodof claim 1 wherein said gas osmotic agent is perfluoropentane and saidmodifier gas is nitrogen.
 24. The method of claim 23 wherein themicrobubble membrane comprises a surfactant.
 25. The method of claim 24wherein said surfactant is selected from the group consisting ofnonionic surfactants, neutral surfactants anionic surfactants, neutralfluorinated surfactants, anionic fluorinated surfactants andcombinations thereof.
 26. The method of claim 24 wherein said surfactantis a fluorinated surfactant.
 27. The method of claim 24 wherein saidsurfactant is a non-Newtonian surfactant.
 28. The method of claim 24wherein said surfactant is selected from the group consisting ofpolyoxypropylene polyoxyethylene copolymers, sugar esters, fattyalcohols, aliphatic amine oxides, hyaluronic acid aliphatic esters,hyaluronic acid aliphatic ester salts, dodecyl poly(ethyleneoxy)ethanol,nonylphenoxy poly(ethyleneoxy)ethanol, hydroxy ethyl starch, hydroxyethyl starch fatty acid esters, dextrans, dextrans fatty acid esters,sorbitol, sorbitol fatty acid esters, gelatin, serum albumins,phospholipids, polyoxyethylene fatty acids esters, polyoxyethylene fattyalcohol ethers, polyoxyethylated sorbitan fatty acid esters, glycerolpolyethylene glycol oxystearate, glycerol polyethylene glycolricinoleate, ethoxylated soybean sterols, ethoxylated castor oil,cholesterol oleic acid, sodium oleate and combinations thereof.
 29. Themethod of claim 1 wherein said gas osmotic agent is perfluorohexane. 30.The method of claim 29 wherein said modifier gas is a fluorocarbonmodifier gas.
 31. The method of claim 29 wherein said modifier gas is anonfluorocarbon modifier gas.
 32. The method of claim 31 wherein saidnonfluorocarbon modifier gas is selected from the group consisting ofnitrogen, oxygen, carbon dioxide and mixtures thereof.
 33. The method ofclaim 31 wherein said nonfluorocarbon modifier gas is air.
 34. Themethod of claim 29 wherein the microbubble membrane comprises asurfactant.
 35. The method of claim 34 wherein said surfactant isselected from the group consisting of nonionic surfactants, neutralsurfactants, anionic surfactants, neutral fluorinated surfactants,anionic fluorinated surfactants and combinations thereof.
 36. The methodof claim 34 wherein said surfactant is selected from the groupconsisting of polyoxypropylene polyoxyethylene copolymers, sugar esters,fatty alcohols, aliphatic amine oxides, hyaluronic acid aliphaticesters, hyaluronic acid aliphatic ester salts, dodecylpoly(ethyleneoxy)ethanol, nonylphenoxy poly(ethyleneoxy)ethanol, hydroxyethyl starch, hydroxy ethyl starch fatty acid esters, dextrans, dextranfatty acid esters, sorbitol, sorbitol fatty acid esters, gelatin, serumalbumins, phospholipids, polyoxyethylene fatty acids esters,polyoxyethylene fatty alcohol ethers, polyoxyethylated sorbitan fattyacid esters, glycerol polyethylene glycol oxystearate, glycerolpolyethylene glycol ricinoleate, ethoxylated soybean sterols,ethoxylated castor oil, cholesterol oleic acid, sodium oleate andcombinations thereof.
 37. The method of claim 34 wherein said surfactantcomprises a mixture of a phospholipid and apolyoxyethylene-polyoxypropylene copolymer.
 38. The method of claim 37wherein said microbubble membrane further comprises a hydroxy ethylstarch.
 39. The method of claim 29 wherein the microbubble membranecomprises a proteinaceous material.
 40. The method of claim 39 whereinsaid proteinaceous material is albumin.
 41. The method of claim 1wherein said gas osmotic agent is perfluorohexane and said modifier gasis nitrogen.
 42. The method of claim 41 wherein the microbubble membranecomprises a surfactant.
 43. The method of claim 42 wherein saidsurfactant is selected from the group consisting of nonionicsurfactants, neutral surfactants, anionic surfactants, neutralfluorinated surfactants, anionic fluorinated surfactants andcombinations thereof.
 44. The method of claim 42 wherein said surfactantis a non-Newtonian surfactant.
 45. The method of claim 42 wherein saidsurfactant is selected from the group consisting of polyoxypropylenepolyoxyethylene copolymers, sugar esters, fatty alcohols, aliphaticamine oxides, hyaluronic acid aliphatic esters, hyaluronic acidaliphatic ester salts, dodecyl poly(ethyleneoxyethanol, nonylphenoxypoly(ethyleneoxy)ethanol, hydroxy ethyl starch, hydroxy ethyl starchfatty acid esters, dextrans, dextran fatty acid esters, sorbitol,sorbitol fatty acid esters, gelatin, serum albumins, phospholipids,polyoxyethylene fatty acids esters, polyoxyethylene fatty alcoholethers, polyoxyethylated sorbitan fatty acid esters, glycerolpolyethylene glycol oxysmate, glycerol polyethylene glycol ricinoleate,ethoxylated soybean sterols, ethoxylated castor oil, cholesterol, oleicacid, sodium oleate and combinations thereof.
 46. The method of claim 42wherein said surfactant comprises at least one phospholipid.
 47. Themethod of claim 42 wherein said surfactant comprises a mixture of aphospholipid and a polyoxyethylene-polyoxypropylene copolymer.
 48. Themethod of claim 42 wherein said microbubble membrane further comprises ahydroxy ethyl starch.
 49. The method of claim 1 wherein saidadministrating step comprises intravenous administration of saidmicrobubble preparation.
 50. The method of claim 1 wherein said object,body part or body cavity to be imaged comprises a vascular system. 51.The method of claim 1 wherein said object, body part or body cavity tobe imaged comprises a perfusion defect.
 52. The method of claim 1wherein said object, body part or body cavity to be imaged comprisesmyocardial tissue.
 53. A method of imaging an object, a body part or abody cavity comprising the steps of: introducing into said object, bodypart or body cavity a microbubble preparation comprising an aqueousmedium having dispersed therein a plurality of osmotically stabilizedmicrobubbles, said microbubbles comprising a generally sphericalmicrobubble membrane containing at least one modifier gas and at leastone gas osmotic agent, wherein said modifier gas and said gas osmoticagent are present in a molar ratio from about 1:100 to about 1,000:1,wherein said ratio is effective to stabilize said microbubblepreparation and wherein said gas osmotic agent comprises the vapor of acompound which is a liquid at 37° C. and 760 Torr, and imaging at leasta portion of said object, body part or body cavity by ultrasound ormagnetic resonance.
 54. The method of claim 53 wherein said gas osmoticagent is selected from the group consisting of perfluorohexane,perfluorocyclohexane, perfluoroheptane, perfluorocycloheptane,perfluoromethylcyclohexane, perfluorodimethylcyclopentane,perfluorotrimethylcyclobutane, perfluorotriethylamine and combinationsthereof.
 55. The method of claim 53 wherein said modifier gas is afluorocarbon gas.
 56. The method of claim 55 wherein said fluorocabonmodifier gas is selected from the group comprising perfluoropropane,perfluorobutane, perfluorocyclobutane, perfluoromethylcyclobutane,perfluoropentane and perfluorocyclopentane.
 57. The method of claim 53wherein said modifier gas is a nonfluorocarbon gas.
 58. The method ofclaim 57 wherein said nonfluorocarbon modifier gas is selected from thegroup consisting of nitrogen, oxygen, carbon dioxide and mixturesthereof.
 59. The method of claim 57 wherein said nonfluorocarbonmodifier gas is air.
 60. The method of claim 53 wherein the microbubblemembrane comprises a surfactant.
 61. The method of claim 60 wherein saidsurfactant is selected from the group consisting of nonionicsurfactants, neutral surfactants, anionic surfactants, neutralfluorinated surfactants, anionic fluorinated surfactants andcombinations thereof.
 62. The method of claim 60 wherein said surfactantis a non-Newtonian surfactant.
 63. The method of claim 60 wherein saidsurfactant is selected from the group consisting of polyoxypropylenepolyoxyethylene copolymers, sugar esters, fatty alcohols, aliphaticamine oxides, hyaluronic acid aliphatic esters, hyaluronic acidaliphatic ester salts, dodecyl poly(ethyleneoxy)ethanol, nonylphenoxypoly(ethyleneoxy)ethanol, hydroxy ethyl starch, hydroxy ethyl starchfatty acid esters, dextrans, dextran fatty acid esters, sorbitol,sorbitol fatty acid ester gelatin, serum albumins, phospholipids,polyoxyethylene fatty acids esters, polyoxyethylene fatty alcoholethers, polyoxyethylated sorbitan fatty acid esters, glycerolpolyethylene glycol oxystearate, glycerol polyethylene glycolricinoleate, ethoxylated soybean sterols, ethoxylated castor oil,cholesterol, oleic acid, sodium oleate and combinations thereof.
 64. Themethod of claim 53 wherein the microbubble membrane comprises aliposome.
 65. The method of claim 53 wherein the microbubble membranecomprises a proteinaceous material.
 66. The method of claim 53 whereinsaid proteinaceous material is albumin.
 67. The method of claim 53wherein said object, body cavity or body part to be imaged comprises avascular systems.
 68. The method of claim 53 wherein said object, bodycavity or body part to be imaged comprises a perfusion defect.
 69. Themethod of claim 53 wherein said object body cavity or body part to beimaged comprises myocardial tissue.
 70. A method of imaging an object, abody part or a body cavity comprising the steps of: providing acontainer having therein microbubble precursor components comprising anaqueous medium, a surfactant, at least one modifier gas that isrelatively soluble in the aqueous medium, and at least one gas osmoticagent that is relatively insoluble in the aqueous medium, wherein saidmodifier gas and said gas osmotic agent are present in a molar ratiofrom about 1:100 to about 1.000:1, wherein said ratio is effective tostabilize a resulting microbubble, with the proviso that said modifiergas is not water vapor, and wherein said microbubble precursorcomponents are adapted to form microbubbles upon the application ofenergy thereto; applying energy to said microbubble precursor componentsto form a microbubble preparation comprising a plurality of microbubblesdispersed in said aqueous medium that are osmotically stabilized whenintroduced into a physiological liquid, in that the gas osmotic agent ispresent in an amount that dilutes the modifier gas sufficiently thatgases ordinarily dissolved in the physiological liquid in vivo seek todiffuse into the bubble with an osmotic pressure sufficient tocounteract the Laplace pressure of the microbubble, said microbubblescomprising a generally spherical microbubble membrane containing saidgas osmotic agent and said modifier gas; introducing at least a portionof said microbubble preparation into said object, body part or bodycavity; and imaging at least a portion of said object, body part or bodycavity by ultrasound or magnetic resonance.
 71. The method of claim 70wherein said gas osmotic agent comprises a fluorocarbon.
 72. The methodof claim 71 wherein said fluorocarbon gas osmotic agent is selected fromthe group consisting of perfluoropropane, perfluorobutane,perfluorocyclobutane, perfluoromethylcyclobutane, perfluoropentane,perfluorocyclopentane, perfluoromethylcyclopentane,perfluorodimethylcyclobutanes, perfluorohexane, perfluorocyclohexane,perfluoroheptane, perfluorocycloheptane, perfluoromethylcyclohexane,perfluorodimethylcyclopentane, perfluorotrimethylcyclobutane,perfluorotriethylamine and combinations thereof.
 73. The method of claim70 wherein said modifier gas is a nonfluorocarbon gas.
 74. The method ofclaim 73 wherein said nonfluorocarbon modifier gas is selected from thegroup consisting of nitrogen, oxygen, carbon dioxide and mixturethereof.
 75. The method of claim 70 wherein said modifier gas is afluorocarbon gas.
 76. The method of claim 70 wherein said surfactant isselected from the group consisting of nonionic surfactants, neutralsurfactants, anionic surfactants, neutral fluorinated surfactants,anionic fluorinated surfactants and combinations thereof.
 77. The methodof claim 70 wherein said gas osmotic agent is perfluoropentane.
 78. Themethod of claim 77 wherein said microbubble precursor componentscomprise perfluoropentane in a liquid state.
 79. The method of claim 78wherein said perfluoropentane is emulsified in said aqueous medium. 80.The method of claim 79 wherein the application of energy reducespressure in said container.
 81. The method of claim 80 wherein theapplication of energy boils said emulsified liquid perfluoropentane toform said microbubble preparation.
 82. The method of claim 77 whereinsaid container is a syringe.
 83. The method of claim 78 wherein saidmodifier gas is nitrogen.
 84. The method of claim 83 wherein saidperfluoropentane is emulsified in said aqueous medium.
 85. The method ofclaim 84 wherein the application of energy reduces pressure in saidcontainer.
 86. The method of claim 85 wherein the application of energyboils sad emulsified liquid perfluoropentane to form said microbubblepreparation.
 87. The method of claim 86 wherein said container is asyringe.
 88. The method of claim 83 wherein said object, body cavity orbody part to be imaged comprises a vascular system.
 89. The method ofclaim 83 wherein said object body cavity or body part to be imagedcomprises a perfusion defect.
 90. The method of claim 83 wherein saidobject, body cavity or body part to be imaged comprises myocardialtissue.
 91. The method of claim 83 wherein said administrating stepcomprises intravenous administration of said microbubble preparation.92. A method of imaging an object, a body part or a body cavitycomprising the steps of: introducing into said object, body part or bodycavity a microbubble preparation, comprising an aqueous medium havingdispersed therein a plurality of microbubbles, said microbubblescomprising a generally spherical microbubble membrane comprisingproteinaceous material containing at least one relatively water-solublemodifier gas and at least one relatively water-insoluble gas osmoticagent in a molar ratio from about 1:100 to about 1.000:1 such that themicrobubbles are osmotically stabilized when introduced into aphysiological liquid, with the proviso that said modifier gas is notwater vapor, and whereby said gas osmotic agent dilutes the modifier gassufficiently that gases ordinarily dissolved in the physiological liquidin vivo seek to diffuse into the bubble with an osmotic pressuresufficient to counteract the Laplace pressure of the microbubble; andimaging at least a portion of said object, body part or body cavity byultrasound or magnetic resonance.
 93. The method of claim 92 whereinsaid gas osmotic agent comprises a fluorocarbon.
 94. The method of claim93 wherein said fluorocarbon gas osmotic agent is selected from thegroup consisting of perfluoropropane, perfluorobutane,perfluorocyclobutane, perfluoromethylcyclobutane, perfluoropentane,perfluorocyclopentane, perfluoromethylcyclopentane,perfluorodimethylcyclobutanes, perfluorohexane, perfluorocyclohexane,perfluoroheptane, perfluorocycloheptane, perfluoromethylcyclohexane,perfluorodimethylcyclopentane, perfluoromethyl cyclobutane,perfluorotriethylamine and combinations thereof.
 95. The method of claim92 wherein said modifier gas is a nonfluorocarbon gas.
 96. The method ofclaim 95 wherein said nonfluorocarbon modifier gas is selected from thegroup consisting of nitrogen, oxygen, carbon dioxide and mixturesthereof.
 97. The method of claim 92 wherein said modifier gas is afluorocarbon gas.
 98. The method of claim 92 wherein the microbubblemembrane further comprises a surfactant.
 99. The method of claim 98wherein said surfactant is selected from the group consisting ofnonionic surfactants, neutral surfactants, anionic surfactants, neutralfluorinated surfactants, anionic fluorinated surfactants andcombinations thereof.
 100. The method of claim 98 wherein saidsurfactant is a fluorinated surfactant.
 101. The method of claim 98wherein said surfactant is selected from the group consisting ofpolyoxypropylene polyoxyethylene copolymers, sugar esters, fattyalcohols, aliphatic amine oxides, hyaluronic acid aliphatic esters,hyaluronic acid aliphatic ester salts, dodecyl poly(ethyleneoxy)ethanol,nonylphenoxy poly(ethyleneoxy)ethanol, hydroxy ethyl starch, hydroxyethyl starch fatty acid esters, dextrans, dextran fatty acid esters,sorbitol, sorbitol fatty acid esters, gelatin, phospholipids,polyoxyethylene fatty acids esters, polyoxyethylene fatty alcoholethers, polyoxyethylated sorbitan fatty acid esters, glycerolpolyethylene glycol oxystearate, glycerol polyethylene glycolricinoleate, ethoxylated soybean sterols, ethoxylated castor oil,cholesterol, oleic acid, sodium oleate and combinations thereof. 102.The method of claim 92 wherein said proteinaceous material is albumin.103. The method of claim 102 wherein said administrating step comprisesintravenous administration of said microbubble preparation.
 104. Themethod of claim 102 wherein said gas osmotic agent comprisesperfluoropropane.
 105. The method of claim 104 wherein said modifier gascomprises nitrogen.
 106. The method of claim 104 wherein said modifiergas comprises oxygen.
 107. The method of claim 104 wherein said modifiergas comprises air.
 108. The method of claim 104 wherein said modifiergas comprises carbon dioxide.
 109. The method of claim 102 wherein saidgas osmotic agent comprises perfluoropropane and said modifier gascomprises nitrogen.
 110. The method of claim 102 wherein said gasosmotic agent comprises perfluoropropane and said modifier gas comprisesoxygen.
 111. The method of claim 102 wherein said gas osmotic agentcomprises perfluoropropane and said modifier gas comprises a mixture ofoxygen and nitrogen.
 112. The method of claim 102 wherein said gasosmotic agent comprises perfluoropropane and said modifier gas comprisesair.
 113. The method of claim 102 wherein said gas osmotic agentcomprises perfluorohexane.
 114. The method of claim 113 wherein saidmodifier gas comprises nitrogen.
 115. The method of claim 113 whereinsaid modifier gas comprises oxygen.
 116. The method of claim 113 whereinsaid modifier gas comprises air.
 117. The method of claim 113 whereinsaid modifier gas comprises carbon dioxide.
 118. The method of claim 113wherein said gas osmotic agent comprises perfluorohexane and saidmodifier gas comprises nitrogen.
 119. The method of claim 102 whereinsaid gas osmotic agent comprises perfluorohexane and said modifier gascomprises oxygen.
 120. The method of claim 112 wherein said gas osmoticagent comprises perfluorohexane and said modifier gas comprises amixture of oxygen and nitrogen.
 121. The method of claim 102 whereinsaid gas osmotic agent comprises perfluorohexane and said modifier gascomprises air.
 122. The method of claim 1, wherein the molar ratio ofsaid modifier gas and said gas osmotic agent is between about 1:100 and1:1.
 123. The method of claim 1, wherein the molar ratio of saidmodifier gas and said gas osmotic agent is between about 1:10 and 1:1.124. The method of claim 124, in which the molar ratio of said modifiergas and said gas osmotic agent is greater than 1:1.
 125. The method ofclaim 125, wherein the modifier gas is a non-fluorocarbon and the gasosmotic agent is a fluorocarbon.
 126. The method of claim 126, whereinsaid plurality of osmotically stabilized microbubbles have a diameterfrom about 1 to 10 μm.
 127. The method of claim 70, wherein the diameterof said plurality of osmotically stabilized microbubbles is about 6 μm.128. The method of claim 120, wherein the molar ratio of said modifiergas and said gas osmotic agent is between about 1:100 and 1:1.
 129. Themethod of claim 70, wherein the molar ratio of said modifier gas andsaid gas osmotic agent is between about 1:10 and 1:1.
 130. The method ofclaim 70, in which the molar ratio of said modifier gas and said gasosmotic agent is greater than 1:1.
 131. The method of claim 130, whereinthe modifier gas is a non-fluorocarbon and the gas osmotic agent is afluorocarbon.
 132. The method of claim 131, wherein said plurality ofosmotically stabilized microbubbles have a diameter from about 1 to 10μm.
 133. The method of claim 132, wherein said plurality of osmoticallystabilized microbubbles have a diameter from about 1 to 10 μm.
 134. Themethod of claim 133, wherein the diameter of said plurality ofosmotically stabilized microbubbles is about 6 μm.
 135. The method ofclaim 92, wherein the molar ratio of said modifier gas and said gasosmotic agent is between about 1:100 and 1:1.
 136. The method of claim92, wherein the molar ratio of said modifier gas and said gas osmoticagent is between about 1:1 0 and 1:1.
 137. The method of claim 82, inwhich the molar ratio of said modifier gas and said gas osmotic agent isgreater than 1:1.
 138. The method of claim 137, wherein the modifier gasis a non-fluorocarbon and the gas osmotic agent is a fluorocarbon. 139.The method of claim 138, wherein said plurality of osmoticallystabilized microbubbles have a diameter from about 1 to 10 μm.
 140. Themethod of claim 139, wherein the diameter of said plurality ofosmotically stabilized microbubbles is about 6 μm.
 141. The method ofclaim 53, wherein the molar ratio of said modifier gas and said gasosmotic agent is between about 1:100 and 1:1.
 142. The method of claim53, wherein the molar ratio of said modifier gas and said gas osmoticagent is between about 1:10 and 1:1.
 143. The method of claim 53, inwhich the molar ratio of said modifier gas and said gas osmotic agent isgreater than 1:1.
 144. The method of claim 143, wherein the modifier gasis a non-fluorocarbon and the gas osmotic agent is a fluorocarbon. 145.The method of claim 144, wherein said plurality of osmoticallystabilized microbubbles have a diameter from about 1 to 10 μm.
 146. Themethod of claim 145, wherein the diameter of said plurality ofosmotically stabilized microbubbles is about 6 μm.